Cell-Free Expression System Having Novel Inorganic Polyphosphate-Based Energy Regeneration

ABSTRACT

The invention relates to an in vitro cell-free expression system incorporating a novel inorganic polyphosphate-based energy regeneration system. In certain embodiments, the invention includes a cell-free expression system where the cellular energy source, ATP, is regenerated from inorganic polyphosphate using a dual enzyme system. In this embodiment, this dual enzyme system may include thermostable Adenosyl Kinase, and/or Polyphosphate Kinase enzymes.

The application is a continuation-in-part of U.S. application Ser. No. 16/309,074, filed Dec. 11, 2018, which claims the benefit of and priority to International Application No. PCT/US18/12121, filed Jan. 2, 2018; which claims the benefit of and priority to U.S. Provisional Patent Application No. 62/440,975, filed Dec. 30, 2016, all of which are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 30, 2020, is named “90125-00210-Sequence-Listing-AF.txt” and is 92.9 Kbytes in size.

TECHNICAL FIELD

The invention relates to a cell-free expression system, the reaction mixture containing all the cell-free reaction components necessary for the in vitro transcription/translation mechanism, amino acids, nucleotides, metabolic components which provide energy and which are necessary for protein synthesis. The invention further relates to an in vitro cell-free expression system incorporating a novel inorganic polyphosphate-based energy regeneration system which may result in higher yields of subject expression products, such as proteins. The invention further relates to a device for in vitro cell-free gene expression. Additionally, the invention relates to a novel inorganic polyphosphate-based energy regeneration system which may be incorporated into diagnostic applications and/or assays.

BACKGROUND

Cell-free expression systems (also known as in vitro transcription/translation, cell-free protein expression, cell-free translation, or cell-free protein synthesis) represent a molecular biology technique that enables researchers to express functional proteins or other target molecules in vitro. Compared to in vivo techniques based on bacterial or tissue culture cells, in vitro protein expression is considerably faster because it does not require gene transfection, cell culture or extensive protein purification. Another advantage of such systems is that often the target protein to be expressed may be toxic to a host cell, or generally incompatible with cellular expression, making in vivo systems impractical if not wholly ineffective vehicles for protein expression.

More specifically, cell-free expression systems generate target molecules and complexes such as RNA species and proteins without using living cells. A typical cell-free expression system may utilize the biological components/machinery found in cellular lysates to generate target molecules from DNA containing one or more target genes. Common components of a typical cell-free expression system reaction may include a cell extract generally derived from a call culture lysate, an energy source such as ATP, a supply of amino acids, cofactors such as magnesium, and the nucleic acid synthesis template with the desired genes, typically in the form of a plasmid synthesis template, or linear expression (or synthesis) template (LET or LST). A cell extract may be obtained by lysing the cell of interest and removing the cell walls, genomic DNA, and other debris through centrifugation or other precipitation methods. The remaining portions of the lysate, or cell extract may contain the necessary cell machinery needed to expresses the target molecule.

A common cell-free expression system involves cell-free protein synthesis (CFPS). To produce one or more proteins of interest, typical CFPS systems harness an ensemble of catalytic components necessary for energy generation and protein synthesis from crude lysates of microbial, plant, or animal cells. Crude lysates contain the necessary elements for DNA to RNA transcription, RNA to protein translation, protein folding, and energy metabolism (e.g., ribosomes, aminoacyl-tRNA synthetases, translation initiation and elongation factors, ribosome release factors, nucleotide recycling enzymes, metabolic enzymes, chaperones, foldases, etc.). Common cell extracts in use today are made from Escherichia coli (ECE), rabbit reticulocytes (RRL), wheat germ (WGE), and insect cells (ICE), and even mammalian cells (MC).

Despite many advantageous aspects of cell-free expression systems, several obstacles have previously limited their use as a protein production technology. These obstacles have included short reaction durations of active protein synthesis, low protein production rates, small reaction scales, a limited ability to correctly fold proteins containing multiple disulfide bonds, and its initial development as a “black-box” science. One significant limitation found in traditional cell-free expression systems is the difficulty in supplying the intense energy and substrate needs of protein synthesis needed for transcription/translation without deleterious concomitant changes in the chemical environment. Furthermore, expensive reagent costs, particularly high energy phosphate chemicals in the form of nucleotides and secondary energy sources, limit the practical uses of such traditional cell-free expression systems.

One of the major limiting factors for in vitro cell-free expression systems is energy regeneration. For example, after initiation of cell-free protein synthesis, production typically continues until one of the substrates (e.g., ATP, cysteine, etc.) is depleted, or byproduct accumulation (e.g., inorganic phosphate) reaches an inhibitory concentration. As such, the inability of any cell-free expression system to efficiently regenerate an appropriate energy source can act as a limiting factor, reducing the overall run-time and ultimate yields of the system. Moreover, it is impracticable, in addition to being prohibitively expensive, to simply add additional ATP into a cell-free expression system as high levels of ATP may actually inhibit protein expression.

To address these issues, traditional cell-free expression systems rely on the addition of supplementary energy sources, primarily in the form of Phosphoenolpyruvate (PEP) and Pyruvatekinase (PK) or Creatinephosphate (CP) and Creatinekinase (CK) or other similar compounds. However, such energy supplementation has significant limitations. First, both substrates PEP and CP have only a functional limited lifetime under in vitro conditions, and their reaction by-product pyruvate is inhibitory to in vitro translation. Second, as noted above, these substrates significantly increase the costs for in vitro applications. Third, both enzymes PK and CK have been demonstrated to have only a limited total turnover number of 3-4 times prior to degradation/inactivity further limiting their usefulness as an energy regeneration source. A protein “turnover” or turnover number (also termed k_(cat)) may be defined as the maximum number of chemical conversions of substrate molecules per second that a single catalytic site will execute for a given enzyme concentration. As a result, because of its poor turnover number, both PK and CK enzymes must be added in significant amounts which limit the run-time of in vitro applications while also increasing costs.

In a more specific example, current E. coli-based cell-free systems primarily rely on creatine phosphate/creatine kinase or on phosphoenolpyruvate (PEP) and Pyruvate Kinase (PK) as the main energy regenerating system, though it is established that PK has a total turnover number in the low single digits and the side-product pyruvate is an inhibitor of the translation reaction. In this manner, not only does the current invention's cellular energy regeneration system teach away from traditional cell-free expression systems, but indeed, represents a demonstrable improvement over traditional cell-free expression systems known in the art.

As a result, there exists a need for a more energetically efficient and robust cell-free expression system that may, for example, have longer reaction duration and increased protein synthesis rates, ultimately producing higher protein yields. Indeed, the foregoing problems regarding the cell-free expression systems, and in particular energy regeneration within these systems, represent a long-felt need for an effective—and economical—solution to the same. While implementing elements may have been available, actual attempts to meet this need may have been lacking to some degree. This may have been due to a failure of those having ordinary skill in the art to fully appreciate or understand the nature of the problems and challenges involved. As a result of this lack of understanding, attempts to meet these long-felt needs may have failed to effectively solve one or more of the problems or challenges here identified. These attempts may even have led away from the technical directions taken by the present inventive technology and may even result in the achievements of the present inventive technology being considered to some degree an unexpected result of the approach taken by some in the field.

As will be discussed in more detail below, the current inventive technology overcomes the limitations of traditional cell-free expression systems, in particular their energy use limitations, while meeting the objectives of a truly energetically efficient and robust in vitro cell-free expression system that results in longer reaction durations and higher product yields.

BRIEF SUMMARY OF THE INVENTION

Generally, the inventive technology relates to cell-free expression systems. One aim of the invention may be to provide methods of cell-free expression that include novel compositions, apparatus and procedures that result in longer reaction times, higher reaction efficiency, and improved reaction stability that ultimately produce higher yields of a desired expression product.

Another aim of the invention may be to provide an improved cell-free expression system, which in one embodiment may include novel systems and methods of energy regeneration. For example, in one preferred embodiment, the inventive technology may include an in vitro cell-free expression system incorporating a novel inorganic polyphosphate-based energy regeneration system. In this preferred embodiment, energy regeneration may be achieved through the addition of inorganic polyphosphate and synergistic high efficiency kinase proteins isolated and purified from select bacterial strains. These kinase proteins may drive the high-efficiency chemical regeneration of the cellular energy source adenosine triphosphate (ATP) within the cell-free expression system. This improved energy regeneration allows for continued activity of the cell-free expression system resulting in higher yields of subject expression products, such as proteins.

In certain embodiments this inorganic polyphosphate-based energy regeneration system may be applied to a variety of cell-free expression systems. For example, in one preferred embodiment the novel energy regeneration system may allow for longer run-time of the system prior to chemical energy depletion and higher yields of expression products, such as proteins, from cell-free translation systems.

Another aim of the inventive technology may relate to energy dependent processes and/or diagnostic assays. Specifically, the inventive technology may include improved in vitro ATP-dependent processes and/or assays having an inorganic polyphosphate-based energy regeneration system. In one preferred embodiment, the inventive energy regeneration system may include an in vitro ATP-dependent protein activity assay having an inorganic polyphosphate-based energy regeneration system. In this embodiment, energy regeneration within the assay may be achieved through the addition of synergistic high-efficiency kinase proteins isolated and purified from select bacterial strains. These kinase proteins may drive the high-efficiency chemical regeneration of ATP in an ATP-dependent assay. This embodiment may allow for longer assay run-time prior to chemical energy depletion, which in-turn may allow for improved assay performance and sensitivity.

Additional aim of the inventive technology may include the identification, isolation, purification and/or modification of RNA polymerase (RNAP) proteins from certain bacterial strains that exhibit increased stability, thermostability and enzymatic activity and/or turnover in cell-free in vitro systems such as those described herein. In one embodiment, a thermostable bacterial RNA polymerase may allow the biosynthesis of messenger RNA (mRNA) from DNA constructs at a higher stability and at higher than traditional temperatures which may aid the unfolding of genomic or Cosmid DNA under, or without the control of, a specific promoter. In one preferred embodiment, this highly stable RNAP may be used to generate expression products in a cell-free expression system, such as in vitro transcription, in vitro cDNA production, and in vitro transcription/translation expression systems. This highly stable RNAP may be used in cell-free expression systems having specific enzymes to regenerate energy sources/substrates such as Uridine triphosphate (UTP), Guanosine triphosphate (GTP), and/or Cytidine triphosphate CTP, as well as an inorganic polyphosphate energy regeneration system as generally described herein. In this embodiment, the increased stability of the RNA polymerase proteins may allow for increased run-time and higher yields in cell-free expression systems as generally described herein. Naturally, reference to certain energy substrates include all forms of such substrates. By way of example, disclosure of GTP includes reference to GTP encompasses guanosine diphosphate (GDP) and guanosine monophosphate (GMP).

Yet another aim of the invention may be to provide one or more genetically modified strains of bacteria that may be optimized for use in cell-free expression systems. Examples of such modifications may include the elimination of certain genes that may, for example, have proteolytic, ribonuclease, and/or sporulation activity to name a few. Additional embodiments may include genetically engineered bacteria that overexpress certain sigma factors that may facilitate promotor recognition in vitro of linear PCR products, as well as resulting in the upregulation of RNAP.

Another aim of the current invention may provide for a novel cell-free expression system growth medium. In certain embodiments, the invention relates to a modified growth medium that may be used in a cell-free expression system. This novel medium may allow optimal bacterial grown without the presence of toxic metal salts and or metal ion chelators.

Additional aims of the inventive technology may become apparent from the detailed disclosure, figures and claims set forth below.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying figures, which are incorporated into and form a part of the specification, illustrate one or more embodiments of the present invention and, together with the description, serve to explain certain aspects of the inventive technology. The drawings are only for the purpose of illustrating one or more preferred embodiments of the invention and are not to be construed as limiting the invention.

FIG. 1a : shows a general diagram of a thermotolerant cell-free expression system fermenter in one embodiment thereof,

FIG. 1b : shows a general diagram of a cell-free expression system fermenter in one embodiment thereof,

FIG. 2a : shows a Gst AdK expression vector in one embodiment thereof;

FIG. 2b : shows a TaqPPK expression vector in one embodiment thereof;

FIG. 3: listing of additional amino acids added to native RNAP protein subunits and RpoD.

FIG. 4: ATPase Luciferase assay demonstrating ATP regeneration utilizing Gst AdK/PPK coupled energy regeneration system. The data specifically showing: row A: 10 uM ATP in 200 ul standard ATPase Luciferase assay in each well; row B: 10 uM ATP, standard PEP/PK conditions (200 uM PEP, 0.02 U/ml PK), row C: 10 uM ATP, ratio PPK/Gst AdK (4:1) total of 1 ug/ml; row D: 10 uM ATP, ratio PPK/Gst AdK (1:1) total of 1 ug/ml; row E: 10 uM ATP, ratio PPK/Gst AdK (1:4) total of 1 ug/ml; columns 1-3: 1 ul (10 mg/ml polyPhosphate); columns 4-6: 3 ul (10 mg/ml polyPhosphate); columns 7-9: 6 ul (10 mg/ml polyPhosphate); columns 10-12: 10 ul (10 mg/ml polyPhosphate). All reactions were recorded continuously for 137 minutes in 96-well plates in a Tecan plate reader (X axis for each well). The total relative luminescence was recorded and is shown for each well (Y axis).

FIG. 5: Under same conditions as for FIG. 4, ATPase Luciferase assay reactions are presented in linear representation of all recorded data points of relative luminescence of Luciferase/D-Luciferin based ATPase assays of ATP, PEP/PK and of the Gst AdK/PPK PPi system for the continuously recorded 392 minutes (X axis). The relative luminescence (same scaling, same reading, same plate) are represented by the Y axis. A separation of all three systems becomes very distinct over time.

FIG. 6: Logarithmic representation of the relative luminescence in Luciferase/D-Luciferin based ATPase assays as an average of all recorded data points (5 independent experiments) of the 3 repeats of: 10 uM ATP in 200 ul assay, 10 uM ATP with PEP/PK conditions (200 uM PEP, 0.02 U/ml PK), 10 uM ATP, ratio PPK/AdK (4:1) total of 1 ug/ml protein and 3 ul (10 mg/ml polyPhosphate). Experiments were recorded for an additional data point after 1008 minutes. All plates stayed in the plate reader to prevent photo bleaching.

FIG. 7: demonstrating the isolation and purification of recombinant proteins Gst AdK and TaqPPK.

FIG. 8: demonstrating ATP energy regeneration system at varied polyphosphate concentrations.

FIG. 9: luciferase assay showing decoupled ATP energy generation reaction products.

FIG. 10: general reaction scheme of the Gst AdK/TaqPPK ATP energy regeneration system as employed in a luciferase assay.

FIG. 11: synergistic Gst AdK and TaqPPK enzymes generating ATP energy out of inorganic polyphosphate and AMP as a substrate source.

FIG. 12: is a comparison of a literature and recombinant PPK protein sequences;

FIG. 13a-b : is a cell-free expression bio-reactor for a cell-free expression system in one embodiment thereof.

FIG. 14: is a cell culture apparatus for generating cell cultures in one embodiment thereof.

MODE(S) FOR CARRYING OUT THE INVENTION(S)

The present invention includes a variety of aspects, which may be combined in different ways. The following descriptions are provided to list elements and describe some of the embodiments of the present invention. These elements are listed with initial embodiments, however it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described systems, techniques, and applications. Further, this description should be understood to support and encompass descriptions and claims of all the various embodiments, systems, techniques, methods, devices, and applications with any number of the disclosed elements, with each element alone, and also with any and all various permutations and combinations of all elements in this or any subsequent application.

The current technology includes a novel inorganic polyphosphate-based energy regeneration having applications for cell-free expression systems including but not limited to: in vitro transcription systems having for example, cell extracts and/or supplemented substrates and enzymes providing for the regulation of UTP, GTP, CTP; in vitro translation systems; in vitro (ATP-dependent) protein activity assay systems; and combined in vitro transcription/translation systems.

In certain embodiments, such energetically synergistic gene products utilized in a cell-free expression system may include, but not be limited to, Adenosyl Kinase (AdK), and/or Polyphosphate Kinase (PPK).

Generally, PPK catalyzes the reversible formation of ATP and inorganic polyphosphate:

ADP+PP _(i) ↔ATP+PP _(i-1)

Generally, Adk catalyzes the interconversion of adenine nucleotides, namely 2 ADP may be converted into one ATP and one AMP.

2ADP↔1ATP+1AMP

In some instances, such energetically synergistic gene products may be generally thermostable, and in some instances, genetically modified for their use in a cell-free expression system. This AdK/PPK coupled energy regulation system may be utilized in a cell-free expression system, as well as other ATP-dependent reactions, diagnostic application and/or assays.

As will be detailed below, the AdK/PPK energy regulation system may include AdK and/or PPK proteins derived from a variety of sources, such as various bacterial species as well as recombinant expressions. As such, while in some embodiments, certain AdK and/or PPK genes or proteins may be specifically identified, however it should be noted that the identification of AdK and/or PPK may generally encompass all AdK and/or PPK genes or proteins and their homologs, paralogs and orthologs as well as all genetically modified versions. Such genetic modification may include mutations, such as one or more point mutations, that may enhance the protein's activity.

In one embodiment, the inventive technology may include the identification and isolation of one or more energetically synergistic gene products from selected bacterial species. Generally referring to FIG. 4, in one preferred embodiment AdK may be cloned from gDNA from Geobacillus stearothermophilus utilizing techniques generally described above. In addition, PPK may be cloned from gDNA from E. coli (ecPPK) (SEQ ID NO. 9), or as will be discuss below, in one preferred embodiment, PPK may be cloned and/or modified from a thermophilic and/or thermotolerant bacteria, such a Thermus aquaticus (TaqPPK).

For example, in one preferred embodiment gDNA from G. stearothermophilus was isolated from an approximately 20 ml overnight culture. The G. stearothermophilus culture may be pelleted and the gDNA isolated using, for example a commercially available kit, such as Macherey-Nagel NuceloSpin Microbial DNA Kit. The gDNA may then be concentrated using ethanol precipitation. This may generally be accomplished by adding a salt and ethanol to a solution containing the gDNA which may precipitate the gDNA which may again be pelleted for example, with a centrifuge and then resuspended in DNase-/RNase-free water. The isolation of gNDA containing a PPK gene in T. aquaticus may be isolated and concentrated by similar means as described above.

Similar to the above referenced description, in one preferred embodiment, both Gst AdK and/or TaqPPK may be cloned using specific primers which may be designed based on the nucleotide sequences of each gene. In this embodiment, both forward and reverse primers may be generated that contain specific restriction sites for the introduction of the subject genes into a selected expression vector(s). In one preferred embodiment, forward and reverse primers may be generated that contain restriction sites to specifically introduce the Gst AdK and/or TaqPPK genes into IBA StarGate Expression Vectors. The subject primers and gDNA from Gst and E. coli may be subject to individual polymerase chain reactions (PCR) to amplify the Gst AdK and/or TaqPPK genes respectively. In one embodiment, individual PCR's for both the Gst AdK and/or ecPPK genes may be performed using standard MasterMix reactions and 50 ng of input gDNA isolated from G. stearothermophilus and E. coli (Table 1) respectively.

In a preferred embodiment, a thermostable PPK enzyme may be derived from the thermophilic T. aquaticus (TaqPPK) and may further be coupled in the inventive cell-free expression system with the Gst AdK (SEQ ID NO. 8) generally identified above. In this embodiment, the PPK literature protein sequence (SEQ ID NO. 10; FIG. 12) was used to back translate into DNA. The sequence was then codon-optimized for E. coli and the gene was subcloned into pET151/D-TOPO for expression (FIG. 2b ). In this embodiment, the subcloned PPK gene, generally identified as TaqPPK, from Thermus aquaticus was genetically modified to contain additional non-naturally occurring amino acids at the N-terminus. These additional non-naturally occurring amino acids include: 1) a 6×His-Tag; 2) a V5 epitope tag and 3) a TEV cleavage site (SEQ ID NO. 11). As noted in Table 1, these three genetic modifications are part of the pET151 backbone and may facilitate expression and purification. TaqPPK was expressed in BL21(DE3) and purified using the methods generally described herein.

In one embodiment, the Gst AdK and TaqPPK gene DNA sequences that have been amplified through the PCR process may be isolated. In one preferred embodiment, PCR reactions may be analyzed on 1% Ethidium Bromide agarose gels and the Gst AdK and/or PPK genes may be isolated by agarose gel extraction and/or PCR purification methods known within the industry such as Macherey-Nagel NucleoSpin Gel and/or PCR Clean-up. These isolated genes may then be inserted into an appropriate delivery vector. For example, in one embodiment the previously generated genes fragments and a selected vector, in this embodiment an appropriate vector (see e.g. FIG. 2a-b respectively), may be digested with selected restriction enzyme(s) and ligated through, for example, a 3:1 T4 DNA ligase reaction which may be available commercially as a Thermo Fisher Scientific Rapid DNA Ligation Kit. It should be noted that for this, and all other embodiments, each gene fragment may be separately ligated into an individual delivery vector and transformed into a select bacterial strain or other appropriate cell. Other embodiments may include ligation of multiple gene fragments into a delivery vector and transformed into a select bacterial strain to be co-expressed.

In this embodiment the ligated Gst AdK and TaqPPK genes and plasmid vector may be individually or collectively transformed into a selected bacterial strain. In a preferred embodiment the individual ligated vector(s) may be transformed into Top10 competent E. coli that may be configured to have a high rate of transformation efficiency. In this embodiment, Top10 competent E. coli may include mutations in the recA gene which may reduce DNA recombination promoting plasmid vector stability and may further include an endA gene knockout which may reduce non-specific endonuclease activity improving plasmid vector yield and quality.

Successfully transformed bacteria may be identified, for example through positive gene detection PCR, or other known methods, and may then be subsequently cultured to generate multiple copies of the plasmid vector within the growing bacterial colony. In a preferred embodiment, the plasmid vector DNA may be isolated and sub-transformed into another select bacterial strain. In a preferred embodiment, the prepared plasmid vector DNA may be sub-transformed into a high expression strain of E. coli, such as BL21(DE3) for optimal protein expression.

In one embodiment the inventive technology may include individual expression of both the Gst AdK and TaqPPK proteins each having a selected molecular tag. In this embodiment, Gst AdK and/or PPK proteins may each be configured to contain a poly-His or His-6 tag, which may be used later for protein purification. In this embodiment, the expressed Gst AdK and/or PPK proteins may be detected and purified because the string of histidine residues bind to several types of immobilized metal ions, including nickel, cobalt and copper, under appropriate buffer conditions.

In one embodiment, a tagged Gst AdK (SEQ ID. NO. 8) and/or TaqPPK (SEQ ID. NO. 11) protein may be individually expressed and purified. In this preferred embodiment, the individually His-6 tagged Gst AdK and/or PPK proteins may be expressed in a BL21(DE3) strain of E. coli. In this embodiment, the BL21(DE3) strain of E. coli may be cultured in NYZ medium, which among other things, may lack lactose which may induce certain proteases that could potentially degrade any expressed proteins, reducing overall yields. Protein expression may be induced at a certain optical density (OD) of bacterial growth. In a preferred embodiment, at approximately OD 0.7, protein expression may be induced by the addition of 0.25 mM isopropyl P-D-1-thiogalactopyranoside (IPTG) for 6-8 hours at 30-32° C. IPTG is a molecular reagent that mimics allolactose, a lactose metabolite that triggers transcription of a lac operon in the plasmid vector DNA, thereby inducing protein expression where the subject gene, in this case the Gst AdK and/or TaqPPK genes respectively, which may be under the control of a lac operator.

In this preferred embodiment, the individual BL21(DE3) cultures may be lysed and a lysate may be prepared and passed through a metal ion-charged resin. The individual lysates may pass through a Ni-charged IMAC resin, where the His-6 tags on the Gst AdK and/or TaqPPK proteins may bind to the immobilized nickel-ion on the resin. Non-binding and non-specific binding proteins may further be removed, in this embodiment through the addition of 5 mM Imidazole, the non-binding and non-specific binding proteins may be removed while the Gst AdK and TaqPPK proteins remain bound to the resin. Again, as noted above, the aforementioned protein purifications steps may be accomplished individually for both Gst AdK and TaqPPK proteins.

The Gst AdK and/or TaqPPK proteins may then be individually eluted by the addition of 300 mM Imidazole or other known methods. The presence of the proteins may be demonstrated through SDS-PAGE and protein purity may be additionally shown by FPLC UV-profiles. In a preferred embodiment the individually purified proteins may be stored in in vitro transcription/translation compatible buffer conditions at a concentration of 13 mg/ml for Gst AdK and 10 mg/ml for PPK at −20° C. for later use.

Further, ATP determination assays may be performed to verify functionality of the Gst AdK and/or PPK proteins in the presence of a polyphosphate. Such an assay may quantify the levels of adenosine triphosphate (ATP) as well as ATP production. ATP (see FIG. 6) is an adenine nucleotide comprised of three phosphate groups esterified to the sugar moiety, found in all living cells. Adenosine triphosphate is involved in energy production for metabolic processes as well as RNA and protein synthesis.

In a preferred embodiment, inorganic polyphosphate, in this case sodium polyphosphate, may be provided as an energy source for the system in an in vitro ATP-dependent system. Isolated and purified Gst AdK and/or TaqPPK may be added to this cell-free expression system. In one preferred embodiment Gst AdK to TaqPPK may be added to a cell-free expression system, or other assay, diagnostic or system, wherein the ratio of Gst AdK to TaqPPK may include higher amounts of Gst AdK as compared to TaqPPK. In in one embodiment, Gst AdK to TaqPPK may be added to this system in an approximate molar ratio of between approximately 3:1-4:1. It should be understood that such ratios are merely exemplary and other ratios are within the scope of this invention. For example, in other embodiments, Gst AdK to TaqPPK may be added to this system in an approximate molar ratio including, but not limited to [See concentration email] 1:1, 2:1, 3:1, 5:1, 5:1, 7:1, 8:1, 9:1, 10:1 and the like.

As generally shown in FIG. 8, in another preferred embodiment, isolated and purified Gst AdK and/or TaqPPK may be added to this cell-free expression system with a quantity of inorganic polyphosphate. In one embodiment, this quantity of inorganic polyphosphate may include an optimal polyphosphate concentration range. In this preferred embodiment, such optimal polyphosphate concentration range being generally defined as the concentration of inorganic polyphosphate (PPi) that maintains the equilibrium of the reaction stable. In this preferred embedment, optimal polyphosphate concentration range may be approximately 0.2-2 mg/ml PPi.

As noted above, PPK can synthesize ADP from polyphosphate and AMP. In this preferred embodiment the coupled action of Gst AdK and PPK, may remove adenosine diphosphate (ADP) from the system by converting two ADP to one ATP and one adenosine monophosphate (AMP):

This reaction may be sufficiently fast enough to drive an equilibrium reaction of PPK towards production of ADP:

In this system, the presence of higher concentrations of AMP may further drive the TaqPPK reaction towards ADP.

Again, as demonstrated in FIGS. 4-6, in ATP determination assays the ATP regeneration rate of the PPK/AdK energy (ATP) regeneration system is significantly higher compared to the traditional PEP/PK energy production systems. In some embodiments the initial ATP conversion rate may be approximately 10-times higher compared to traditional PEP/PK systems under comparable conditions. Additionally, the Gst AdK and PPK further demonstrated in FIGS. 4-6, may have a higher total turnover number which results in a detectable ATP regeneration for an extended period, which in this embodiment was up to 12 hours.

Incorporation of TaqPPK may further result in a detectable ATP regeneration in excess of 12 hours. Such ATP regeneration turnover being significantly higher than traditional in vitro ATP regeneration systems described above. In other embodiments, Gst AdK and/or TaqPPK may include improved salt tolerance allowing continued enzymatic activity and/or turnover in salt concentrations that would be inhibitory for other AdK proteins. As such, it can be seen how Gst AdK and TaqPPK, (as well as AdK and/or PPK and their homologs derived from various other bacteria, including but not limited to, thermophilic and/or thermotolerant microbial strains) in the presence of a polyphosphate may work synergistically within a cell-free expression system to regenerate the cellular energy source ATP:

As noted above, in certain embodiments ribosomes, and ribosomal factors may be included in a cell-free expression system. Notably, ribosomes, and ribosomal factors are dependent on guanosine triphosphate (GTP) for their energy, not ATP. As such, in certain preferred embodiments, a cell-free expression system may include one or more nucleotide kinase enzymes NDPK that may convert ATP into GTP which may be utilized by the ribosomes, and ribosomal factors in a cell-free expression system incorporating such factors. As used herein, the term “NDPK” means an enzyme having the activity of the enzyme classified as EC 2.7.4.6, namely the transfer of the 7-phosphate group of a nucleoside triphosphate (NiTP) to a nucleoside diphosphate (N₂DP) via a ping-pong mechanism where each step is part of a reversible process, such that the multistep equilibrium is of the following form.

NDPK+NTP↔NDPK˜NTP↔NDPK−P˜NDP↔NDPK−P+NDP

A ping-pong mechanism integrates phosphorylation of a histidine residue by transferring terminal phosphate group (7-phosphate) from ATP to NDP P-phosphate in order to produce an NTP, and NDPK catalyzes such reversible reactions. NTP phosphorylates a histidine, which in turn phosphorylates NDP. NDPK are involved in the synthesis of nucleoside triphosphates (NTP), such as guanosine triphosphate (GTP), cytidine triphosphate (CTP) and uridine triphosphate (UTP), thymidine triphosphate (TTP). The ping-pong mechanism can further be visualized as:

In one preferred embodiment, a quantity of NDPK may be isolated and included in a cell-free expression system, and preferably a cell-free expression system configured to produced peptides. In this embodiment, the NDPK may convert ATP to GTP in an equilibrium. In a preferred embodiment, a thermostable NDPK, or NDPK derived from a thermophile may be used, for example, in one embodiment a thermostable NDPK derived from Thermus thermophilus according to SEQ ID NO. SEQ ID NO. 23 may be used to convert ATP to GTP in a cell-free expression system. In this embodiment, the NDPK may only need to be added at low concentrations, for example 1/20 to 1/5 of the concentration of ADK, and preferably 1/10 of the concentration of Adk. (See Table 2, and FIG. 16)

Certain embodiments of the inventive technology may include cell-free expression systems having an improved energy regeneration system. In one preferred embodiment, a cell-free expression system, such as an in vitro transcription and/or translation system may be configured to include an inorganic polyphosphate energy regeneration capability that may further include thermostable RNA polymerase (RNAP) from select bacterial strains.

In one preferred embodiment, RNAP subunits alpha (SEQ ID NO. 1), beta (SEQ ID NO. 2), beta′ and or beta′ (opt) (SEQ ID NO. 3 and SEQ ID NO. 4 respectively), delta (SEQ ID NO. 5) and omega (SEQ ID NO. 6) from the bacterial species Geobacillus stearothermophilus were identified, cloned, purified and prepared for use in a cell-free protein expression system. It should be noted that G. stearothermophilus is a gram positive thermophilic bacteria characterized by an inner cell membrane and a thick cell wall. G. stearothermophilus is a rod shaped anaerobe found in thermophilic habitats like thermal vents or hot springs.

An RNAP from G. stearothermophilus (Gst RNAP) may be identified, cloned, expressed and then purified as generally described herein. In this embodiment the Gst RNAP may be more stable than, for example, standard RNAP from non-extremophile bacteria such as E. coli. The Gst RNAP may have improved thermostability, as well as improved kinetic stability, such that it may remain viable to catalyze the production of mRNA in an in vitro system for a longer period of time than other RNAP variants used in traditional cell-free expression systems.

In one preferred embodiment, a thermostable Gst RNAP may be cloned and transformed into a high expression E. coli strain. This thermostable Gst RNAP may be cloned, isolated and purified then introduced into a cell-free expression system in the presence of a target genetic material, such as plasmid and/or linear DNA containing one or more select genes, and allowed to produce mRNA transcripts of the select genes. In one embodiment, these mRNA transcripts may be introduced to, or combined with a bacterial or other cellular extract. The extract may contain the necessary elements for DNA to RNA transcription, RNA to protein translation, protein folding, and energy metabolism. As noted above, in this embodiment, isolated and purified TaqPPK and Gst AdK may be added to, for example, a cell extract of a cell-free expression system, as well as a quantity of one or more polyphosphates during protein translation to provide a mechanism of improved ATP regeneration.

In this preferred embodiment, translation of the target mRNA transcripts may generate one or more select protein(s) while synergistic activity of the TaqPPK, Gst AdK and polyphosphate provide enhanced ATP regeneration, allowing longer run-times of the cell-free expression system, at possibly higher temperatures if desired, and having a higher turnover number resulting in overall higher protein yields. In this embodiment, the presence of a highly stable Gst RNAP allows for longer run-times for the production of mRNA transcripts, at possibly higher temperatures without being denatured and having a higher turnover number resulting in higher mRNA transcripts and ultimately protein yields within the cell-free expression system.

The invention further relates to an improved in vitro transcription/translation system, generally referred to as a cell-free expression system. In one embodiment, this cell-free expression system may include one or more genetically modified strains of thermophilic or thermotolerant bacteria that may be developed for use in cell-free extract preparation. In a preferred embodiment, a genetically modified strain of a thermophilic bacteria, such as a Geobacillus strain, may be developed and used for cell-free (CF) extract preparation and for in vitro transcription/translation. Geobacillus strains generally exhibit a high growth temperature (52-55° C.) compared to E. coli (35-37° C.). This thermal differential may allow for higher protein stability and toleration of higher protein densities in CF extract preparations. Further, Geobacillus strains' thermophilic attributes may allow for increased stability for other enzyme-, and enzymatic functions, such as, but not limited to: amino acid-synthases; ribosomes; DNA-dependent RNA polymerase; sigma factor(s); and ATP regenerating and glycolytic enzymes.

Bacteria from the genus Geobacillae are also advantageous for CF extract preparations. Being firmicutes, they are 02-tolerant or strictly aerobic, under specific conditions. Handling, fermentation and extract preparations of anaerobic thermophiles may be significantly more complex, costlier, and have additional uncertainties related to enzyme stability due to oxidizing conditions of the atmosphere. Moreover, Geobacilli are known to have no known health implications to humans, and as such, are suitable to produce clinical material from a CF system.

With respect to the invention's genetically modified Geobacillus strain, it was determined that the ribosome binding site (RBS) was predicted and demonstrated by genetic analysis to be identical to, or comparable to, the standard RBS of expression constructs for E. coli protein expression strains, allowing the inventive technology in a preferred embodiment to preserve and utilize existing genetic constructs.

In another preferred embodiment, the invention's genetically modified Geobacillus strain demonstrated favorable coverage of tRNA codon usage. In one embodiment, bioinformatic analysis was performed using a proprietary software tool with tRNA population distribution to identify strains that cover all or most all potential codons to prevent stalling of the in vitro translation reaction due to rare or not covered tRNA codons. Extracts of this Geobacillus strain may be also utilize purified E. coli tRNAs, which are recognized by the strain's amino acid synthases. This may allow complete and almost equally distributed codon coverage. This embodiment takes the natural unequal distribution of tRNA codon usage of varying strains, such as Geobacillus stearothermophilus, and Thermophilus aquaticus into account in favor of a thermodynamically balanced distribution.

As generally discussed above, in one preferred embodiment, a genetically modified bacterial strain was developed for use in a cell-free expression system. In a preferred embodiment, a Geobacillus strain was genetically modified to remove and/or reduce the expression of certain enzymes, and to constitutively overproduce enzymes during the fermentation process to facilitate the use of CF extract preparations. (This genetically modified strain may sometimes be generally referred to as “Geobacillus CF”). In this embodiment, a Geobacilli was genetically modified to knock-out the strong protease OmpT-homologue, RNaseI, and the DNA-methylation dependent DNase. In this embodiment, the genetic knock-out modifications were accomplished through the selective removal of the transcription control of these genes from the bacterial genome. Other knock-out methods may also be employed, as they are generally known and understood in the art. In this embodiment, the activity of the culture density-dependent sporulation operon was reduced by genetic modification (knocked down) to slow the sporulation process and to increase the bacterial density in culture allowing for higher efficiency and improved CF extract yield.

Again, as generally discussed above, in certain embodiments a genetically modified bacterial strain was developed to overexpress sigma factor, RpoD, to recognize a specific promoter for favored in vitro transcription from linear PCR products. In a preferred embodiment, a Geobacillus CF was genetically modified to overexpress sigma factor, RpoD during the fermentation of the strain. This genetically modified overexpression of RpoD elevated expression of this sigma factor above the natural cellular concentration of sigma 70, which in turn resulted in an increased concentration of the bacterial RNAP in the genetically modified Geobacillus strain. Finally, the genetically modified bacterial Geobacillus strain exhibits growth rates comparable to E. coli protein expression strains known in the art. Additional advantages of using the genetically modified Geobacilli strain for CF extract preparation include: 1) a high growth temperature (approximately 52-55° C.) which significantly reduces the potential contamination of the culture; and 2) “spent” fermentation media are simple and non-hazardous, reducing the need for waste treatment, as it would be required for anaerobic thermophiles.

In additional embodiment, a cell extract may be generated from a number of different micro-organisms. For example, cell extracts may be generated from Escherichia coli (ECE), rabbit reticulocytes (RRL), wheat germ (WGE), and insect cells (ICE), and even mammalian cells (MC). In additional embodiment, cell-extracts from microalgae may be generated. Such an embodiment may be advantageous, as bacteria do not provide any mechanism to recognize and remove introns. On the other hand, microalgae, like nanochloropsis, have introns and the cellular machinery remove them—which would naturally be a constituent of an microalgae cell extract making the current invention applicable for the production of plant proteins and other expression products.

In vitro cell-free expression, as used herein, refers to the cell-free synthesis of polypeptides in a reaction mixture or solution comprising biological extracts and/or defined cell-free reaction components. The reaction mix may comprise a template, or genetic template, for production of the macromolecule, e.g. DNA, mRNA, etc.; monomers for the macromolecule to be synthesized, e.g. amino acids, nucleotides, etc., and such co-factors, enzymes and other reagents that are necessary for the synthesis, e.g. ribosomes, tRNA, polymerases, transcriptional factors, etc. The cell-free synthesis reaction, and/or cellular adenosine triphosphate (ATP) energy regeneration system component,s may be performed/added as batch, continuous flow, or semi-continuous flow.

As noted above, the current invention may be applicable to a number of assays, and other diagnostic applications. For example, in one embodiment, the addition of the dual enzymes Adk/PPK, along with PPi and AMP may allow for ATP regeneration within any ATP energy dependent assay or diagnostic application, such as a luciferase assay. In one preferred embodiment, a first quantity of Gst AdK, and a first quantity of TaqPPK, may be added to an ATP dependent assay or diagnostic, along with a first quantity of PPi and a first quantity AMP. In this preferred embodiment, cellular ATP energy regeneration may be achieved allowing for improve assay and/or diagnostic run-time, output, sensitivity, as well as loser cost due to the inexpensive nature of the components compared to traditional energy supplementation techniques. Each of the above referenced components may further be supplemented over a specified time-course.

Examples of such ATP-dependent application may include, but not be limited to energy-dependent light reactions or enzyme reactions could benefit; Epimerases, Dehydratases, NADPH-Hydrate-Dehydratases, Cellulases, Chromatin-remodeling-enzymes, ATP-dependent peptide ligases, DNA ligation, and traditional cell-free expression systems. The inventive system may be applied in the generation of: Synthestases; Phosphorylation processes; CoA dependent enzymes (detergents, flavor industry); Deaminoacide amides (Deala-Deala-Ligases) As noted above, in one embodiment of the present invention a cell-free expression system having a coupled Adk/PPK energy regeneration system may include the addition of inorganic polyphosphate (PPi). In another embodiment, a cell-free expression system having a coupled Adk/PPK energy regeneration system may include the addition of a first quantity of PPi, while still other embodiments may include the addition of a plurality of quantities of PPi. In this embodiment, a plurality of quantities of PPi may be added at a pre-determined time interval.

In one preferred embodiment, a cell-free expression system having a coupled AdK/PPK energy regeneration system may include a concentration range sufficient to maintain the energy regulation reaction at the approximate state of equilibrium, and/or produce the maximum, or desired level of ATP. In yet another preferred embodiment, a cell-free expression system having a coupled AdK/PPK energy regeneration system may include concentration approximately between 0.1 and 50 mg/ml PPi or higher. In yet another preferred embodiment, a cell-free expression system having a coupled AdK/PPK energy regeneration system may include a concentration range sufficient to maintain the energy regulation reaction at an approximate state of equilibrium, such range being approximately between 0.3-4 mg/ml PPi.

In yet another preferred embodiment, a cell-free expression system having a coupled AdK/PPK energy regeneration system may include a concentration range sufficient to maintain the energy regulation reaction at an approximate state of equilibrium, such range being approximately between 0.2-2 mg/ml PPi. In yet another preferred embodiment, a cell-free expression system having a coupled AdK/PPK energy regeneration system may include a concentration range sufficient to maintain the energy regulation reaction at the approximate state of equilibrium, such range being approximately between 0.5-0.6 mg/ml PPi. Naturally, all such ratios may be scaled up or down as may be required based on the size, duration, and energy requirements of a cell-free expression reaction. In some other embodiments, a functional range may also include no additional PPi, or 0 mg/ml.

Generally referring to FIGS. 13a-b , in one embodiment the invention relates to a cell-free expression bioreactor (22) (see elements 10-20, including a Reactor vessel, a feeding solution reservoir (10) an Inlet (11), a Stir bar (12), a plurality of seals (13), a Dialysis membrane (14), a Reaction vessel (15), a Lid (16), a Feeding solution (17), an Outlet with groove for air bubble removal (18) a Reaction mixture (19), and a plurality of Tighteners (20)). It should be noted that such a bioreactor is exemplary only as multiple cell-free systems may be implemented with this invention. The inventive bio-reactor may allow cell-free expression at higher than traditional temperatures and may be configured to run as a batch, continuous or semi-continuous, from for example a feeder reaction solution.

Again, referring to FIG. 14, in this embodiment the invention may further include a cell-free culture apparatus (21) (see elements 1-9, including a Recirculation bath (1), and Air compressor; alternate air/gas supply (2), a fermentation vessel (3), and Insulated water bath (4), and Air stone (5), and Air supply line (6), a Water return (7), a Hot water supply (8), and a Sterile filtered air vent (9)). This cell culture apparatus may be configured to culture, in certain preferred embodiment thermophilic bacteria. A fermentation vessel may be removable and separately autoclavable in a preferred embodiment. Additionally, this cell-free culture apparatus (21) may be configured to accommodate the growth of aerobic as well as anaerobic with organisms. Moreover, both the cell-free expression bioreactor (22) and cell-free culture apparatus (21) may accommodate a variety of cell cultures, such a microalgae, plant cells and the like.

It should be noted that the following cell-free protein expression system is exemplary in nature and different configurations and systems may be incorporated with the aforementioned inorganic polyphosphate energy regeneration system. In addition, the novel inorganic polyphosphate energy regeneration system may be adapted to a variety of known cell-free expression systems and may further be configured into an adaptable kit.

Because this invention involves production of genetically altered organisms and involves recombinant DNA techniques, the following definitions are provided to assist in describing this invention. The terms “isolated”, “purified”, or “biologically pure” as used herein, refer to material that is substantially or essentially free from components that normally accompany the material in its native state or when the material is produced. In an exemplary embodiment, purity and homogeneity are determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A nucleic acid or particular bacteria that are the predominant species present in a preparation is substantially purified. In an exemplary embodiment, the term “purified” denotes that a nucleic acid or protein that gives rise to essentially one band in an electrophoretic gel. Typically, isolated nucleic acids or proteins have a level of purity expressed as a range. The lower end of the range of purity for the component is about 60%, about 70% or about 80% and the upper end of the range of purity is about 70%, about 80%, about 90% or more than about 90%.

The term “nucleic acid” as used herein refers to a polymer of ribonucleotides or deoxyribonucleotides. Typically, “nucleic acid” polymers occur in either single- or doublestranded form but are also known to form structures comprising three or more strands. The term “nucleic acid” includes naturally occurring nucleic acid polymers as well as nucleic acids comprising known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Exemplary analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). “DNA”, “RNA”, “polynucleotides”, “polynucleotide sequence”, “oligonucleotide”, “nucleotide”, “nucleic acid”, “nucleic acid molecule”, “nucleic acid sequence”, “nucleic acid fragment”, and “isolated nucleic acid fragment” are used interchangeably herein. For nucleic acids, sizes are given in either kilobases (kb) or base pairs (bp). Estimates are typically derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or amino acid residue numbers. Proteins sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.

Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), the complementary (or complement) sequence, and the reverse complement sequence, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (see e.g., Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). In addition to the degenerate nature of the nucleotide codons which encode amino acids, alterations in a polynucleotide that result in the production of a chemically equivalent amino acid at a given site, but do not affect the functional properties of the encoded polypeptide, are well known in the art. “Conservative amino acid substitutions” are those substitutions that are predicted to interfere least with the properties of the reference polypeptide. In other words, conservative amino acid substitutions substantially conserve the structure and the function of the reference protein. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine or histidine, can also be expected to produce a functionally equivalent protein or polypeptide. Exemplary conservative amino acid substitutions are known by those of ordinary skill in the art. Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.

Homology (e.g., percent homology, sequence identity+sequence similarity) can be determined using any homology comparison software computing a pairwise sequence alignment. As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are to have “sequence similarity” or “similarity”. Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Henikoff S and Henikoff J G. [Amino acid substitution matrices from protein blocks. Proc. Natl. Acad. Sci. U.S.A. 1992, 89(22): 10915-9],

According to a specific embodiment, the homolog sequences are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or even identical to the sequences (nucleic acid or amino acid sequences) provided herein. Homolog sequences of SEQ ID Nos 1-22 of between 50%-99% may be included in certain embodiments of the present invention.

The term “primer,” as used herein, refers to an oligonucleotide capable of acting as a point of initiation of DNA synthesis under suitable conditions. Such conditions include those in which synthesis of a primer extension product complementary to a nucleic acid strand is induced in the presence of four different nucleoside triphosphates and an agent for extension (for example, a DNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature.

A primer is preferably a single-stranded DNA. The appropriate length of a primer depends on the intended use of the primer but typically ranges from about 6 to about 225 nucleotides, including intermediate ranges, such as from 15 to 35 nucleotides, from 18 to 75 nucleotides and from 25 to 150 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template nucleic acid but must be sufficiently complementary to hybridize with the template. The design of suitable primers for the amplification of a given target sequence is well known in the art and described in the literature cited herein.

As used herein, a “polymerase” refers to an enzyme that catalyzes the polymerization of nucleotides. “DNA polymerase” catalyzes the polymerization of deoxyribonucleotides. Known DNA polymerases include, for example, Pyrococcus furiosus (Pfu) DNA polymerase, E. coli DNA polymerase I, T7 DNA polymerase and Thermus aquaticus (Taq) DNA polymerase, among others. “RNA polymerase” catalyzes the polymerization of ribonucleotides. The foregoing examples of DNA polymerases are also known as DNA-dependent DNA polymerases.

RNA-dependent DNA polymerases also fall within the scope of DNA polymerases. Reverse transcriptase, which includes viral polymerases encoded by retroviruses, is an example of an RNA-dependent DNA polymerase. Known examples of RNA polymerase (“RNAP”) include, for example, T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase and E. coli RNA polymerase, among others. The foregoing examples of RNA polymerases are also known as DNA-dependent RNA polymerase. The polymerase activity of any of the above enzymes can be determined by means well known in the art.

The term “reaction mixture,” or “cell-free reaction mixture” as used herein, refers to a solution containing reagents necessary to carry out a given reaction. A cell-free expression system “reaction mixture” or “reaction solution” typically contains a crude or partially-purified extract, (such as from a bacteria, plant cell, microalgae, fungi, or mammalian cell) nucleotide translation template, and a suitable reaction buffer for promoting cell-free protein synthesis from the translation template. In some aspects, the CF reaction mixture can include an exogenous RNA translation template. In other aspects, the CF reaction mixture can include a DNA expression template encoding an open reading frame operably linked to a promoter element for a DNA-dependent RNA polymerase. In these other aspects, the CF reaction mixture can also include a DNA-dependent RNA polymerase to direct transcription of an RNA translation template encoding the open reading frame. In these other aspects, additional NTPs and divalent cation cofactor can be included in the CF reaction mixture. A reaction mixture is referred to as complete if it contains all reagents necessary to enable the reaction, and incomplete if it contains only a subset of the necessary reagents. It will be understood by one of ordinary skill in the art that reaction components are routinely stored as separate solutions, each containing a subset of the total components, for reasons of convenience, storage stability, or to allow for application-dependent adjustment of the component concentrations, and that reaction components are combined prior to the reaction to create a complete reaction mixture. Furthermore, it will be understood by one of ordinary skill in the art that reaction components are packaged separately for commercialization and that useful commercial kits may contain any subset of the reaction components of the invention. Moreover, those of ordinary skill will understand that some components in a reaction mixture, while utilized in certain embodiments, are not necessary to generate cell-free expression products.

The term “cell-free expression products” may be any biological product produced through a cell-free expression system.

The term “about” or “approximately” means within a statistically meaningful range of a value or values such as a stated concentration, length, molecular weight, pH, time frame, temperature, pressure or volume. Such a value or range can be within an order of magnitude, typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The allowable variation encompassed by “about” or “approximately” will depend upon the particular system under study. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and includes the endpoint boundaries defining the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

The term “recombinant” or “genetically modified” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, organism, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein, or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells may express genes that are not found within the native (nonrecombinant or wild-type) form of the cell or express native genes that are otherwise abnormally expressed, over-expressed, under-expressed or not expressed at all.

As used herein, the term “transformation” or “genetically modified” refers to the transfer of one or more nucleic acid molecule(s) into a cell. A microorganism is “transformed” or “genetically modified” by a nucleic acid molecule transduced into the bacteria or cell or organism when the nucleic acid molecule becomes stably replicated. As used herein, the term “transformation” or “genetically modified” encompasses all techniques by which a nucleic acid molecule can be introduced into a cell or organism, such as a bacteria.

As used herein, the term “promoter” refers to a region of DNA that may be upstream from the start of transcription, and that may be involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A promoter may be operably linked to a coding sequence for expression in a cell, or a promoter may be operably linked to a nucleotide sequence encoding a signal sequence which may be operably linked to a coding sequence for expression in a cell.

The term, “operably linked,” when used in reference to a regulatory sequence and a coding sequence, means that the regulatory sequence affects the expression of the linked coding sequence. “Regulatory sequences,” or “control elements,” refer to nucleotide sequences that influence the timing and level/amount of transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters; translation leader sequences; introns; enhancers; stem-loop structures; repressor or binding sequences; termination sequences; polyadenylation recognition sequences; etc. Particular regulatory sequences may be located upstream and/or downstream of a coding sequence operably linked thereto. Also, particular regulatory sequences operably linked to a coding sequence may be located on the associated complementary strand of a double-stranded nucleic acid molecule.

As used herein, the term “genome” refers to chromosomal DNA found within the nucleus of a cell, and also refers to organelle DNA found within subcellular components of the cell. The term “genome” as it applies to bacteria refers to both the chromosome and plasmids within the bacterial cell. In some embodiments of the invention, a DNA molecule may be introduced into a bacterium such that the DNA molecule is integrated into the genome of the bacterium. In these and further embodiments, the DNA molecule may be either chromosomally-integrated or located as or in a stable plasmid.

The term “gene” or “sequence” refers to a coding region operably joined to appropriate regulatory sequences capable of regulating the expression of the gene product (e.g., a polypeptide or a functional RNA) in some manner. A gene includes untranslated regulatory regions of DNA (e.g., promoters, enhancers, repressors, etc.) preceding (up-stream) and following (down-stream) the coding region (open reading frame, ORF) as well as, where applicable, intervening sequences (i.e., introns) between individual coding regions (i.e., exons). The term “structural gene” as used herein is intended to mean a DNA sequence that is transcribed into mRNA which is then translated into a sequence of amino acids characteristic of a specific polypeptide.

The term “expression,” as used herein, or “expression of a coding sequence” (for example, a gene or a transgene) refers to the process by which the coded information of a nucleic acid transcriptional unit (including, e.g., genomic DNA or cDNA) is converted into an operational, non-operational, or structural part of a cell, often including the synthesis of a protein. Gene expression can be influenced by external signals; for example, exposure of a cell, tissue, or organism to an agent that increases or decreases gene expression. Expression of a gene can also be regulated anywhere in the pathway from DNA to RNA to protein. Regulation of gene expression occurs, for example, through controls acting on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization, or degradation of specific protein molecules after they have been made, or by combinations thereof. Gene expression can be measured at the RNA level or the protein level by any method known in the art, including, without limitation, Northern blot, RT-PCR, Western blot, or in vitro, in situ, or in vivo protein activity assay(s).

The term “vector” refers to some means by which DNA, RNA, a protein, or polypeptide can be introduced into a host. The polynucleotides, protein, and polypeptide which are to be introduced into a host can be therapeutic or prophylactic in nature; can encode or be an antigen; can be regulatory in nature, etc. There are various types of vectors including virus, plasmid, bacteriophages, cosmids, and bacteria.

An “expression vector” is nucleic acid capable of replicating in a selected host cell or organism. An expression vector can replicate as an autonomous structure, or alternatively can integrate, in whole or in part, into the host cell chromosomes or the nucleic acids of an organelle, or it is used as a shuttle for delivering foreign DNA to cells, and thus replicate along with the host cell genome. Thus, an expression vector are polynucleotides capable of replicating in a selected host cell, organelle, or organism, e.g., a plasmid, virus, artificial chromosome, nucleic acid fragment, and for which certain genes on the expression vector (including genes of interest) are transcribed and translated into a polypeptide or protein within the cell, organelle or organism; or any suitable construct known in the art, which comprises an “expression cassette.” In contrast, as described in the examples herein, a “cassette” is a polynucleotide containing a section of an expression vector of this invention. The use of the cassettes assists in the assembly of the expression vectors. An expression vector is a replicon, such as plasmid, phage, virus, chimeric virus, or cosmid, and which contains the desired polynucleotide sequence operably linked to the expression control sequence(s).

The terms “expression product” as it relates to a protein expressed in a cell-free expression system as generally described herein, are used interchangeably and refer generally to any peptide or protein having more than about 5 amino acids. The polypeptides may be homologous to, or may be exogenous, meaning that they are heterologous, i.e., foreign, to the organism from which the cell-free extract is derived, such as a human protein, plant protein, viral protein, yeast protein, etc., produced in the cell-free extract.

A “cell-free extract” or “lysate” may be derived from a variety of organisms and/or cells, including bacteria, thermophilic bacteria, thermotolerant bacteria, archaea, firmicutes, fungi, algae, microalgae, plant cell cultures, and plant suspension cultures.

As used herein the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the culture” includes reference to one or more cultures and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs unless clearly indicated otherwise.

The invention now being generally described will be more readily understood by reference to the following examples, which are included merely for the purposes of illustration of certain aspects of the embodiments of the present invention. The examples are not intended to limit the invention, as one of skill in the art would recognize from the above teachings and the following examples that other techniques and methods can satisfy the claims and can be employed without departing from the scope of the claimed invention. Indeed, while this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

EXAMPLES Example 1: Generation of Thermostable Gst RNAP from G. stearothermophilus

In this preferred embodiment, the present inventors have demonstrated that RNA polymerase from G. stearothermophilus (Gst RNAP) may be cloned from genomic DNA (gDNA) isolated from a G. stearothermophilus culture. In this embodiment, approximately 20 ml G. stearothermophilus may be grown in an overnight culture and then pelleted, for example with a centrifuge. The gDNA may then be isolated, in this instance by using commercially available gDNA purification kits, such as a Macherey-Nagel NuceloSpin Microbial DNA Kit. The gDNA may then be concentrated using ethanol precipitation. This may generally be accomplished by adding a salt and ethanol to a solution containing the gDNA which may precipitate the gDNA which may again be pelleted for example with a centrifuge and then resuspended in DNase-/RNase-free water.

In this preferred embodiment, the subunits of the Gst RNAP may be cloned using specific primers which may be designed based on the nucleotide sequences of each gene. Forward and reverse primers that contain specific restriction sites to introduce the subunit genes into an expression vector may be utilized. In one preferred embodiment, forward and reverse primers that contain restriction sites to specifically introduce the subunit genes into an IBA StarGate Expression Vector(s) may be generated. Next the subject primers and gDNA may be subject to a polymerase chain reaction (PCR) to amplify the Gst RNAP subunit genes. In one preferred embodiment PCRs may be performed using standard MasterMix reactions and 50 ng of input gDNA isolated from G. stearothermophilus.

As noted above, it may be preferred to select a Gst RNAP subunit to act as an optimized gene sequence, perhaps due to the presence of in internal restriction site. Generally referring to FIG. 3, Gst RNAP subunit beta′ (opt) (SEQ ID NO. 4) may be identified and cloned in a manner similar to that described above. Also referring to FIG. 3, a transcription factor, in this case RpoD (SEQ ID NO. 7), may be used from a commercially available codon optimized gene synthesis (Invitrogen) and cloned directly into an expression vector.

In one embodiment, the Gst RNAP subunit DNA sequences that have been amplified through the PCR process may be isolated. In one preferred embodiment, PCR reactions may be analyzed on 1% Ethidium Bromide agarose gels and the subunit fragments may be isolated by agarose gel extraction and/or PCR purification methods known within the industry such as Macherey-Nagel NucleoSpin Gel and PCR Clean-up. These gene fragments may then be inserted into an appropriate delivery vector. For example, in one embodiment, the previously generated gene fragments and a selected vector, in this embodiment a commercially available StarGate vector (see e.g. FIG. 2), may be digested with selected restriction enzyme(s) and ligated through, for example, a 3:1 T4 DNA ligase reaction which may be available commercially as a Thermo Fisher Scientific Rapid DNA Ligation Kit.

Next, in this embodiment the ligated gene fragments and plasmid vector may be transformed into a selected bacterial strain. In a preferred embodiment the ligated vector(s) may be transformed into the bacteria Escherichia coli, in particular Top10 competent E. coli that may be configured to have a high rate of transformation efficiency. In this embodiment, Top10 competent E. coli may include mutations in the recA gene which may reduce DNA recombination promoting plasmid vector stability and may further include an endA gene knockout which may reduce non-specific endonuclease activity improving plasmid vector yield and quality.

Successfully transformed bacteria may be identified, for example, through positive gene detection PCR, or other known methods, and may then be subsequently cultured to generate multiple copies of the plasmid vector within the growing bacterial colony. In a preferred embodiment, the plasmid vector DNA may be isolated and sub-transformed into another select bacterial strain. In a preferred embodiment, the prepared plasmid vector DNA may be sub-transformed into a high expression strain of E. coli, such as BL21(DE3) for optimal protein expression.

In one embodiment, one or more expressed RNAP subunit proteins may be configured with a molecular tag. Referring now to FIG. 3, the RNAP beta′ (opt) subunit may be configured to contain a poly-His or His-6 tag, which may be used later for protein purification. In this embodiment, the expressed His-tagged RNAP beta′ (opt) subunit can be purified and detected easily because the string of histidine residues binds to several types of immobilized metal ions, including nickel, cobalt and copper, under appropriate buffer conditions.

In one embodiment, a tagged Gst RNAP may be expressed and purified. In this preferred embodiment, the His-6 tagged beta′ (opt) subunit may be expressed in a BL21(DE3) strain of E. coli. In this embodiment, the BL21(DE3) strain of E. coli may be cultured in NYZ medium, which among other things, may lack lactose that may induce certain proteases that could potentially degrade any expressed proteins, reducing overall yields. Protein expression may be induced at a certain optical density (OD) of bacterial growth. In a preferred embodiment, at OD 0.7, protein expression may be induced by the addition of 0.25 mM isopropyl P-D-1-thiogalactopyranoside (IPTG) for 6-8 hours at 30-32° C. IPTG is a molecular reagent that mimics allolactose, a lactose metabolite that triggers transcription of a lac operon in the plasmid vector DNA, thereby inducing protein expression of the Gst RNAP subunit gene under the control of a lac operator (see FIG. 2a ).

In this preferred embodiment the BL21(DE3) cultures may be lysed and a lysate prepared and passed through a metal ion-charged resin. This lysate may pass through a Ni-charged IMAC resin, where the His-6 tag on the beta′ (opt) subunit may bind to the immobilized nickel ion on the resin. Non-binding and non-specific binding proteins may further be removed in this embodiment through the addition of 5 mM Imidazole, while the beta′ (opt) subunit remains bound to the resin.

Next, expression and isolation of the remaining Gst RNAP subunits may occur. In a preferred embodiment, a combined lysate of protein expression strains for the remaining Gst RNAP subunits may be prepared and incubated with beta′ (opt)-loaded resin for approximately 2-3 hours. Non-binding and non-specific binding proteins may again be removed through the addition of 5 mM Imidazole. The expressed subunits may assemble to form a RNAP complex bound to the resin through the beta′ (opt) subunit, and the assembled Gst RNAP may then be eluted by the addition of 300 mM Imidazole or other known methods. The assembly of the Gst RNAP may be demonstrated by SDS-PAGE and further stored in in vitro transcription/translation compatible buffer conditions at a concentration of 10 mg/ml at −20° C. for later use. Further, protein activity assays may be performed to verify assembly and functionality of the GSt RNAP complex. The presence of mRNA transcripts being indicative of successful Gst RNAP isolation and assembly. In one embodiment, a 25 μl assay may be performed comprising:

-   -   4 mM each ribonuclease tri-phosphate (rNTP);     -   250 ng DNA;     -   2 μl Gst RNAP;     -   20 U RNase inhibitor; and     -   5 μl 5× reaction buffer

Example 2: Gst AdK/TagPPK Isolation and Purification

The present inventors demonstrated the isolation and purification of Gst AdK and TaqPPK, which purified proteins were utilized in subsequent assays as described below. Specifically, Gst AdK and TaqPPK were expressed and isolated utilizing the methods as generally described herein. Referring to FIG. 7, the purified, recombinant proteins were analyzed on a 4-20% SDS PAGE. The gel being stained with coomassie dye.

Specifically, aliquots of the purified proteins were analyzed on SDS PAGE for purity and correct size. A previously test-purified PPK protein (PPK) was used as an additional size marker for the lot purification of the recombinant TaqPPK (PPK fractions 1-3). All fractions eluted from the purification column are demonstrated as pure and show the correct size of around 74 kDa. The purified Gst AdK shows no contaminant proteins and has the correct size of around 25 kDa.

Example 3: Coupled AdK/PPK ATP Energy Regeneration

The present inventors demonstrated the coupled AdK/PPK_ATP regeneration capability of the invention through a commercially available ATP determination assay. Here, the present inventors employed Life technologies, D-Luciferin, A22066 to verify functionality of the Gst AdK and/or PPK proteins, (in this embodiment derived from E. coli), in ATP regeneration.

In this embodiment, in addition to the standard 200 μl assay components, the following materials may be added to the assay, with the results being provided in FIGS. 4-6.

-   -   i) 10 μM ATP     -   ii) 5 μl Gst AdK     -   iii) 5 μl PPK derived from E. coli (exemplary only)     -   iv) 50 μl polyphosphate (1 mg/ml stock solution)

As demonstrated in FIGS. 4-6, these data generally demonstrate the enhanced ATP regeneration capabilities of the invention's AdK/PPK-PPi ATP energy regeneration system, and its improvement over ATP alone, as well as traditional PEP/PK energy supplementation. Specifically, the data clearly separate: lowest: ATP, middle: PEP/PK, and upper: AdK/PPK-PPi

Example 4: AdK/PPK Energy Regeneration at Variable Polyphosphate Concentrations

The present inventors demonstrated coupled Gst AdK/TaqPPK energy regeneration may be achieved at variable polyphosphate concentrations through a commercially available ATP determination assay. Specifically, a standard reaction setup was varied in the amount of added PPi. The luciferase signal (RLU) was measured over time (min).

As demonstrated in FIG. 8, one concentration range that maintained the equilibrium of the reaction was determined to be between approximately 0.2-2 mg/ml PPi. Other concentration ranges were determined to maintain the equilibrium of the reaction. Specifically, such ranges were determined to be between 0.1-4 mg/ml PPi, and/or 0.5-0.6 mg/ml, and/or 0.1-7 mg/ml. As demonstrated in FIG. 8, some additional ATP regeneration was found within the functional ranges, including, but not limited to approximately 0-48 mg/ml and higher.

In one embodiment, a standard reaction setup for 100 μl luciferase assay may include:

standard final conc. 20× buffer 5 1× 100 mM DTT 1 1 mM DTT 3 mg/mL D-luciferin 5 150 μg/ml luciferin 5 mg/mL luciferase 0.2 10 mg/ml luciferase 25 mM AMP 10 2.5 mM AMP 0.34 mg/ml TaqPPK 67 228 μg/ml PPK 13 mg/ml Gst AdK 0.2 .226 mg/ml AdK 100 mg/ml Sodium 0.5 0.5 mg/ml polyphosphate dH2O 11.1 TOTAL 100

Again, as demonstrated in FIG. 8, ATP regeneration was sustained for more than five hours, at which point the measurements were stopped. As shown, the present inventors demonstrate that higher or lower inputs of PPi may disturb a stable reaction cycle of Gst AdK/TaqPPK and, while functional, do not result in the highest levels of continuous energy regeneration. Naturally such ranges are approximations and not intended to be limiting.

Example 5: Decoupled AdK/PPK ATP Energy Regeneration Out of AMP/PPi

The present inventors demonstrated that the Gst AdK/TaqPPK system can generate energy (ATP) out of inexpensive materials, such as PPi and AMP as a source. To further confirm this novel ATP regeneration system, the present inventors decoupled the reaction of Gst AdK/TaqPPK from the luciferase assay. Specifically, the present inventors first generated ATP from AMP/PPi using purified Gst AdK and TaqPPK ATP proteins, purified the reactions and used samples to run a luciferase assay—which as noted above, requires ATP to emit light.

As shown in FIG. 9, the present inventors show that only a standard setup with all reaction components provided sufficient ATP for a luciferase light-emitting reaction. The controls which had individual compounds/enzymes or combinations thereof missing, did not provide ATP for a successful luciferase reaction. Since the ATP energy was not regenerated in this assay, the luciferase signal drops over time and reaches almost background after around seven minutes at which point all previously generated ATP was used up.

Example 6: Generation of ATP Energy Out of AMP/PPi and its Regeneration by Gst AdK/TaqPPK

The present inventors demonstrated the generation of ATP energy out of AMP and PPI and that the ATP regeneration, through at least one embodiment of the Gst AdK/TaqPPK energy regeneration system. Specifically, the present inventors performed a luciferase assay, replacing the usually needed ATP by AMP and added the energy regeneration system components, namely Gst AdK, TaqPPK, and PPi). In control experiments, the present inventors omitted each component and combinations thereof.

The reaction scheme of the Gst AdK/TaqPPK ATP energy regeneration system, in the context of an ATP-using luciferase reaction to emit light, is generally demonstrated in FIG. 10. In a first step, ADP is generated in an irreversible reaction by PPK using AMP and PPi as a source. ADP can be converted into ATP by PPK and AdK, both in reversible reactions keeping the amounts of AMP, ADP and ATP balanced. ATP is then used by luciferase to convert luciferin and emit light.

As shown in FIG. 11, the standard energy regeneration system components showed an increasing luciferase signal (RLU) after enough ATP is made to support the light emitting reaction. The signal increases over time until it reaches a maximum after around 7 hours. Control reactions with the energy regeneration system missing do not show any RLU. A such, the present inventors demonstrate that it is the coupled, or synergistic Gst AdK and TaqPPK enzymes generating energy (ATP) out of inexpensive polyphosphate and AMP as a substrate source. The present inventors were able to measure a light emitting reaction for approximately 20 hours (not shown), further demonstrating the robust energy regeneration possible from their novel system.

Example 7: Cell-Free Extract Preparation

A defined, non-toxic, non-hazardous medium based on genetically modified organism-free (GMO-free), purified soy peptone and animal-sourced materials free (ASM-free) casein tryptone was developed by the present inventors for fermentation of the genetically modified Geobacillus strains. This medium does not require the addition of potentially toxic metal salts or metal ion chelators, (e.g., Trinitrileacetic acid, etc.), for a productive fermentation of the genetically modified Geobacillus strains. The fermentation temperature range may be set and kept stable at approximately 52-55° C., although larger temperature ranges are also contemplated and enabled. The culture may be maintained at an approximate pH of 7.2-7.6, although larger pH ranges are also contemplated and enabled. The addition of 10 mM of HEPES buffer, may be added as needed. Non-toxic, silicone-based anti-foaming agents may be added, as needed, for a controlled fermentation process.

Ribosomal RNA (rRNA) was isolated from growing cultures at different stages of the fermentation process and their concentration determined as a measure of ribosome concentration per culture unit. A maximum concentration of rRNA was observed after 6-7 hours of growth in fresh media. In some embodiments, cultures may be grown in multiple phases with increasing volumes, and the final 7.5 L. culture is inoculated to OD₆₀₀ 0.5 and grown for 6-8 hours to a final OD₆₀₀ of 2.5-2.8 at 52-55° C. and pH 7.2-7.6.

The lysate is prepared after a single freeze/thaw cycle overnight at −80° C. from collected washed cells (3×). After the freeze/thaw cycle, the cells are re-suspended in a HEPES buffer saline buffer containing RNase and protease inhibitors (1.25:1 cells to buffer ratio) and are disrupted by ultrasound at 2-4° C. using an established protocol for less than 5 mins (10 sec “on”, 30 sec “off” cycling). The used lysis buffer, CF-L1, is non-toxic, non-hazardous. Particulates are removed from the lysate by centrifugation (12,000 g, 4° C., 10 mins), and dialyzed against a non-toxic storage buffer, CF-St, which is similar in composition to the reaction buffer CF-1 but does contain 20% v/v glycerol and no protease inhibitors. A 30-minute run-off reaction (shaking in a closed tube at 250 rpm and at 30° C.) and additional centrifugation (same conditions) to further clarify the extract, is not usually required. Potential surviving bacteria and/or bacterial and fungal contaminations are determined by streaking samples of the lysate and of the pelleted debris on agar plates of mLB/CS-medium, NB, ISP2, blood-brain agar and are incubated at 30° C. and 55° C. for two days. The cell disruption rate is consistently above 99.95% (<1 survivor per 200 μL of resuspended debris and <1 colony forming unit (CFU) per mL of lysate). In one embodiment, processing of a 7.5 L fermenter may produce approximately 30 ml of extract. Naturally, based on the operator's desire and/or need, these amounts/volumes may be scaled up and/or down accordingly.

Total RNA extractions of cleared lysates may be used to determine the ribosome concentration. The total protein concentration of the lysate is determined by UV/Vis spectroscopy and is consistently in the range of 150-160 mg/mL. Aliquots of the lysate are stored at −80° C. and may be stable for 6 months or longer, as demonstrated by determining the stability of the extract (no precipitation) and consistent in vitro transcription/translation rates of a Cas9-sfGFP construct (determined by UV/Vis absorption and fluorescence measurements). The lysate and in vitro transcription/translation reactions may be maintained in an approximate range of pH 7.8-8.0. The salt concentrations for the CF reaction conditions may be variable. Each lysate batch may be evaluated by the in vitro transcription/translation activity of a Cas9-sfGFP linear construct (production of a larger protein with consistent folding) and of a luciferase construct (enzymatic activity).

Example 8: Cell-Free Extracts Generated Utilizing Genetically Modified Geobacillus Strains

As noted above, the present inventors observed that the over-expression of the sigma factor RpoD (from Geobacillus) up-regulated the bacterial RNA polymerase (RNAP) in bacterial cultures of E. coli and Geobacillus. Recombinant genetic constructs were generated to constitutively express RpoD_(x), an engineered version, in Geobacilli. The functional reliability of the genetic construct and the protein expression levels were determined and are monitored using a sfGFP construct transformed into the same Geobacillus strain and are monitored by 510 nm fluorescence readings of prepared lysates.

The present inventors further utilized the RhIII promoter for in vitro transcription together with the bacterial RNAP. A recombinant modified RhIII promoter for tighter RpoD/promoter recognition may be utilized in some embodiments. Alternatively, a T7 promoter can be used, if, for example, a T7 RNAP polymerase is added to the CF reaction. In some embodiments, a proprietary genetically modified T7 RNAP (T7x) having one or more single point mutations compared to the native RNAP from bacteriophage T7 may be utilized. Such point mutations may allow a faster melting of the double stranded DNA and increase the complex stability of the RNAP with the single stranded template DNA. In this embodiment, a T7x may cause significantly fewer incomplete transcripts and produce consistently higher mRNA concentrations compared to commercial T7-RNAP. The present inventors have demonstrated that both RNAPs perform reliably, with reproducible and predictable results, for constructs up to 45,000 bases in length.

Example 9: In Vitro Cell-Free Expression System Having Novel ATP Energy Regeneration System

As detailed above, the invention relates, in certain embodiments to a cell-free expression system where the cellular energy source, ATP, is regenerated from inorganic polyphosphate using a dual enzyme system. One enzyme for the system was developed utilizing a thermostable polyphosphate kinase (PPK) from Thermophilus aquaticus (TaqPPK), after demonstrating that a polyphosphate hydrolyzing activity was observed for a homologue enzyme cloned and produced from E. coli (ecPPK or PPK). Other than the reported PPK activity, isolated from Vibrio cholerae, TaqPPK and ecPPK preferably generate ADP from AMP and the hydrolysed inorganic polyphosphates (iPP_(i)). For the second enzymatic step, two molecules of ADP are then rearranged by a thermostable Adenosine Kinase (AdK) to form one unit of ATP and AMP.

This system has proven to operate effectively for at least 32 hours, as determined by measurements of luciferin luminescence after adding luciferase to a solution of 10 nM AMP, 150 pg/ml luciferin, 1 mg/ml iPP_(i) and catalytic amounts of the enzymes. A total enzyme turnover number was determined for the Gst AdK/TaqPPK pair of at least 300. This enzymatic loop may remain active for at least 72 hours and up to at least 1 week or longer. In one embodiment, the ATP concentration in the feeding solution may be maintained at a stable 1.5 mM ATP. The present inventors have further demonstrated that TaqPPK may have a maximum total turnover number of approximately 3,000, hence, allowing the further reduction of the protein concentration of TaqPPK in the inventive cell-free expression system. This may prevent or minimize random hydrolysis of polyphosphate, which causes a feedback inhibition by accumulating pyrophosphate to the cell-free reactions, while producing/maintaining the desired levels of ATP.

Such novel energy regeneration characteristics are important, as current E. coli-based cell-free systems primarily rely on creatine phosphate/creatine kinase or on phosphoenolpyruvate (PEP) and Pyruvate Kinase (PK) as the main energy regenerating system, though it is established that PK has a total turnover number in the low single digits and the side-product pyruvate is an inhibitor of the translation reaction. In this manner, not only does the current invention teach away from such traditional cell-free expression systems, but indeed, represents a demonstrable improvement that traditional cell-free expression systems known in the art.

The redox potential of the present inventor's cell-free expression system may be stabilized by a Sorbitol-dehydrogenase (SDH). In one embodiment, this SDH may be thermostable (tSDH) (such t designation being applicable generally to other molecules referenced herein). Enzymatic assays using the 340 nm absorption for NADH with purified protein samples demonstrated that the forward conversation from sorbitol and NAD/NAD(P) to fructose and NADH/NAD(P)H is 8 times faster compared to the back reaction. The present inventors cloned and produced this genetically modified thermostable enzyme from Geobacillus stearothermophilus, which was also optimized for expression in E. coli as previously described here (Gst SDH), which may be added to the feeding solution along with 200 mM sorbitol. The reaction product also provides an additional energy resource via glycolysis and does not accumulate in the reaction mix.

In certain embodiments, the cell-free expression system may include an initial volume of 5 ml for the cell-free reaction volume, and 80 ml for the feeding solution to ensure a continuous exchange and homogeneous reaction conditions in the vessels. Cell-free expression systems between 0.5 ml and 2 liter reaction volumes and higher may further be contemplated.

Example 10: Cell-Free Expression System Reaction Set Up

In one embodiment, the CF system may be adjusted and stabilized in the range of pH 7.9-8.0 with a TRIS-acetate buffer (CF-1) in the feeding and reaction compartments. The final concentration of the lysate in the reaction vessel may be between 100-110 mg/ml total protein, while template DNA (alternatively also together as a pre-run RNA/RNAP reaction) are added together with cofactors, amino acids and nucleotides to the lysate. The feeding solution contains equal concentrations of the nucleotides and all natural amino acids. Cofactors, sorbitol and the aforementioned enzymes are added as well. A detailed list of materials is provided below and may generally be referred to as cell-free reaction components. Additionally, the general components of the dual-enzyme ATP regeneration system may be included. Such components in one preferred embodiment may include but are not limited to, a quantity of Gst AdK, a quantity TaqPPK, and/or a quantity of PPi, and/or a quantity of AMP. Additional embodiments may include a quantity of Gst SDH and sorbitol.

This exemplary cell-free reaction may run in an incubator at approximately 35-37° C., while the reactor is agitated at approximately ˜300 rpm for 16 hours. In certain embodiments, this reaction setup or reaction mixture of the combined in vitro transcription/translation reactions may be suitable to produce the target protein for more than 3 days. In one exemplary embodiment, the inventive cell-free expression system may provide the stability to produce proteins for more than 36 hours.

In on specific embodiment, cell-free reaction components may include, but not be limited to:

amino acids

polyphosphate

Tris-Acetate

Mg(OAc)₂

K⁺-glutamate

amino-acetate

NaCl

KCl

MgCl₂

DTT

octyl-b-glycoside

NAD (converted to NADH in the system)

NADP (converted to NADPH in the system)

sorbitol

FADH (regenerated by NADH/FADH cellular processes)

ATP

GTP, UTP, CTP each

CoA

PLP

SAM

In one specific embodiment, cell-free reaction components may include, but not be limited to:

2 mM of each natural amino acid

1 mg/ml polyphosphate

5 mM Tris-Acetate

4 mM Mg(OAc)₂

12 mM K*-glutamate

1 mM amino-acetate

100 mM NaCl

10 mM KCl

5 mM MgCl₂

0.1 mM DTT

0.2% octyl-b-glycoside

0.8 mM NAD (converted to NADH in the system)

0.4 mM NADP (converted to NADPH in the system)

200 mM sorbitol

0.5 mM FADH (regenerated by NADH/FADH cellular processes)

1.5 mM ATP

1 mM GTP, UTP, CTP each

1 mM CoA

2 mM PLP

0.2 mM SAM

All amounts, concentrations, volumes and ratios of any cell-free reaction component are exemplary only, as they may be variable based on reaction volume, duration, or expression product to be produced, and other variables. Cell-free reaction components may further include, but not be limited to, all other components necessary for transcription/translation mechanism, amino acids, nucleotides, metabolic components which provide energy and which are necessary for the synthesis, whether provided by the cell extract, and or added to the cell-extract. Cell-free reaction components may further include a quantity of tRNA specific for the strain of bacteria, algae, microalgae archaea, fungi and/or firmicute that may be employed. For example, in this embodiment described above, natural Geobacillus tRNAs may be added. Additionally, cell-free reaction components may further include a quality of tRNA that may overcome stalling of translation due to rare codons. For example, in some embodiments, tRNAs from E. coli mre600 may be included in the cell-free reaction to overcome stalling of translation due to rare codons.

Example 11: High-Efficiency Generation of Clostridium tetani E88 Tentoxilysin by In Vitro Cell-Free Expression

The present inventors demonstrate, as an exemplary embodiment, the in vitro expression of Tentoxilysin (tetanus neurotoxin) utilizing at least one embodiment of the inventive cell-free expression system that utilizes a coupled Gst AdK/TaqPPK ATP energy regeneration as generally described herein.

Tentoxilysin (TetNT) is naturally produced as a 150 kDa single chain protoprotein. TetNT coordinates with a Zn²⁺ cation at the endopeptidase motif, M₂₃₂HELIHVL₂₃₉, which enables the proteolytic cleavage of the full-length polypeptide between A₄₅₇ and S₄₅₈. The selective oxidation of two Cysteine residues (C₄₃₉ and C₄₆₇) forms a disulfide bridge between both chains and allows the formation of the active tetanus toxin protein complex consistent of a short chain, M₁-A₄₅₇, and a long chain, S₄₅₈-A₁₃₁₉. This protein is then selectively exported by the natural producing strains of the Cl. tetani family.

TetNT is an internalizing Zn²⁺-dependent endopeptidase, which hydrolyzes Synaptobrevin-2 between Gln₇₆ and Phe₇₇ via a toxin/ganglioside receptor complex formation. The hydrolysis of the Synaptobrevin-2 receptors on synaptic vesicles prevents the release of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) into the synaptic cleft. This ultimately causes rigid muscular spasms, besides other symptoms, of the patient described as Tetanus. The tetX gene was localized in the Cl. tetani E88 genome and was previously further encoded on the E. coli protein expression plasmid CTC_p60. The gene product was previously cloned and expressed, as a heterologous protein to confirm the protein activity and to conduct high-resolution structural protein studies. A potential N-acetyl-b-neuraminic acid modifying activity of TetNT was previously described and known in the art as well.

According to FDA regulations, currently tetX toxoid, suitable for human vaccination, is derived from active Cl. tetani culture. The completely folded and exported toxin is removed from the active culture by flocculation resulting from the addition of aluminum sulfate and ammonium sulfate. The extracted toxin is subsequently inactivated by the addition of formaldehyde to gain toxoid suitable for vaccinations. FDA suggests quality control measures in the form of SDS-PAGE, FPLC or HPLC profiles and purifications, which are currently not required.

The established isolation and preparation protocol currently known in the art carries risks. These risks have been acknowledged by the FDA, as the agency recommends tetanus vaccinations for children of 7 years of age and older, and adults only. The vaccination carries a 5% risk of infections with Cl. tetani. The biggest challenges for fermentation-based production of Tet toxoid are the control of formaldehyde concentrations to ensure the complete deactivation of the toxin and the control of the toxoid concentration in the final product resulting in 40% of all vaccinations being ineffective.

As an exemplary embodiment, these challenges and the production limitations can be addressed by replacing the fermentation process with the inventive in vitro transcription/translation based production method. The carry-over of spores and any other colony formers is not existent and ensures full sterility of the Active Pharmaceutical Ingredient (API). The total protein concentration is monitored during the whole process and the final product may be controlled by standard laboratory methods.

The present inventors generated a genetic construct for TetNT expression. In this embodiment, the genetic construct was designed for gene synthesis and, specifically, to be fed into the in vitro transcription/translation, or cell-free expression reaction as a linear PCR template. The use of linear DNA in the present cell-free expression system may be preferable in some embodiments as it cannot be transferred, as it is unable to recombine, replicate or be transfected. The linear genetic construct may include the following basic configuration: ((promoter)-(RBS)−(startexpression product cleavage site-detection tag+purification tag)(stop). In one specific embodiment, this configuration may include: (promoter)-(RBS)−(start)(TetNT TEV mCherry H6)(stop).

In this embodiment, the promoter may include: a RhIII promotor sequence (SEQ ID NO. 18), or alternatively a T7 promotor sequence (SEQ ID NO. 19). An RBS (ribosomal binding site) may be derived from a T7 gene phage consistent with Geobacillus sequences (SEQ ID NO. 20) (naturally a number of promotors may be utilized apart from these exemplary examples). A recombinant TetNT gene may include SEQ ID NO. 21. In this recombinant TetNT gene, the stop sequence is removed. Finally, a TEVsite, mCherry, His6 may include SEQ ID NO. 22, again, with the stop removed from the mCherry. As should be noted, while provided as separate sequences, the genetic construct may be presented as a single linear PCR fragment of DNA generally described.

In this instance, due to safety and security concerns, the DNA coding for the full-length tetanus toxin, in a preferred embodiment, gene synthesis may proceed as two separate circular constructs, which will be recombined in the laboratory to a single plasmid for amplification purposes. For example, a first construct coding for the TetNT light chain (SEQ ID NO. 12) with the autocatalytic protease site (SEQ ID NO. 13) and disulfide bridge forming cysteines. A second construct will code for the TetNT heavy chain (SEQ ID NO. 14), containing the partially integrated TEV site (SEQ ID NO. 15) and mCherry-His6 (SEQ ID NO. 16). While the recombinant protein is presented as individual genetic constructs and amino acids sequences, such sequences may be part of a single construct that ultimately produce a single linear polypeptide (SEQ ID NO. 17), which may correspond to a polynucleotide sequence, as a PCR amplified linear DNA construct. Additional embodiments may include disparate constructs that may form a single linear polypeptide.

The present inventors demonstrate expression of a full-length 150 kDa pro-toxin by cell-free protein synthesis further utilizing the previously described dual enzyme ATP regeneration system. In this specific embodiment, expression of a full-length 150 kDa pro-toxin by cell-free protein synthesis may occur in the 5 ml reactor according to described conditions. The reaction progress may be monitored by daylight fluorescence of tagged mCherry with an excitation wavelength of 587 nm and red-shifted fluorescence. After no further increase in protein concentration is observed, the content of the reaction vessel will be transferred into a 50 ml reaction tube. The reaction times and protein concentrations attained can be determined continuously.

A 10 mM HEPES buffer pH 8.0, containing lactose, 200 mM NaCl, 1 mM DTT, may be added to dilute the reaction mix 10-fold. 1-2 mL of a Ni-IDA resin will be added to this solution and incubated at approximately 20-25° C. for 1 hour with limited agitation. The binding capacity of this resin may be sufficient for up to 60 mg of His6-tagged protein. After the His6-tagged pro-toxin is bound to the resin, the resin will be collected, and the supernatant wash buffer removed. Fluorescence will immediately confirm that no TetNT pro-toxin is present in the supernatant.

The collected protein-charged resin may be washed with 50 ml of HEPES buffer at pH 8.0, containing lactose, 200 mM NaCl, and incubated at ambient temperature, with no reducing agent present. Under exposure to O₂ in various concentrations (from 2% to atmospheric) and for various times and in absence of reducing agents, the spontaneous folding/refolding, oxidative formation of the native disulfide bridges, will be initiated within the long chain of TetNT and in between the long and short chain of TetNT.

Subsequent to the folding/refolding, ZnCl₂ may be added to a final concentration of 2 mM to the protein solution. This activates the internal autoproteolytical activity to cleave the peptide bond between the long chain and short chain of the resin-adsorbed TetNT. An incubation time of approximately 2 hours at 20-25° C. may be used initially. To this point the toxin is inert, as the engineered modifications with mCherry and a His6-tag will prevent its activity due to steric hinderance of the recognition and binding sites of its natural target. Furthermore, the bead size of the resin increases the total particle size significantly and reduces the risk of the protein being aerosolized or absorbed. The easy to detect fluorescence of mCherry helps to keep track of the protein and to prevent unwanted exposure or unintended disposal of potentially toxic material at this stage. The buffer may be exchanged to remove all incompletely folded toxin fragments that did not form disulfide bridges and will remove access ZnCl₂ from the solution.

A purified His6-tagged TEV protease may be added to the reaction mix and the sample is incubated for several hours at approximately 20-25° C. The TEV protease may be adsorbed to the resin as well and will remain bound, while the activated Tetanus toxin is released from the resin by cleaving the TEV protease site. This process can be monitored, by the quenching of the mCherry fluorescence, due to the cleavage of the TEV site overlapping with the missing N-terminus of mCherry and the resulting partial unfolding of the fluorescing protein. After no fluorescence can be detected, the resin, which is still charged with unfolded mCherry-His6 and TEV-His6 protease, is collected and the supernatant should contain only pure TetNT in a sterile and desired buffer.

The supernatant may be tested by the present inventors with a proprietary anti-Tet Fab, to confirm the correct protein folding in ELISA and western blot assays, while SDS-PAGE and FPLC profile confirm purity of the sample. After the protein concentration is determined the required amount of formaldehyde can be added to gain the inactivated toxoid. Animal assays to determine effective doses to confirm the degree of correctly folded and active protein in the final sample may further be conducted. Naturally, as described above, this protocol is exemplary in nature. Those of ordinary skill in the art will recognize that such protocols may be similarly employed, without undue experimentation to produce similar proteins from other Clostridia as well as other proteins of interest.

REFERENCES

The following references are hereby incorporated in their entirety by reference:

-   [1] Carlson, Erik D. et al. “Cell-Free Protein Synthesis:     Applications Come of Age.” Biotechnology advances 30.5 (2012):     1185-1194. PMC. Web. 1 Jan. 2018.

SEQUENCE LISTINGS

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. In some cases, single letter designations for amino acids is provide in this specification, while three-letter designations have been provided in the electronically submitted format. All prior sequence data disclosed in U.S. Provisional Application No. 62/440,975 is hereby incorporated by reference.

TABLE 1 Sequence Listings SEQ ID NO. 1 SGSSMIEIEKPKIETVELSEDAKYGKFVVEPLERGYGTTLGNSLRRILLSSLPGAAVTSVQIDG VLHEFSTIDGVVEDVTAIILNIKKLALKIYSDEEKTLEIDVQGEGVVTAADITHDSDVEILNPD LHIATLAEGGRLRMRMTAKRGRGYVPAEANKREDQPIGVIPIDSIYTPVSRVSYQVENTRVGQV TDYDKLTIDVWTDGSIGPKEAISLGAKILTEHLNIFVGLTDEAQNAEIMVEKEDDQKEKVLEMT IEELDLSVRSYNCLKRAGINTVQELTQKTEEDMMKVRNLGRKSLEEVKAKLAELGLSLRKDDGR RAA SEQ ID NO. 2 SGSSMTGRLVQYGRHRQRRSYARISEVLELPNLIEIQTSSYQWFLDEGLREMFREISPIEDFSG NLSLEFIDYSLGEPKYTVEEAKERDVTYAAPLRVKVRLINKETGEVKEQDVFMGDFPLMTETGT FlINGAERVIVSRLVRSPSVYYSDKVDKNGKRGYSATVIPNRGAWLEYETDAKDVVYVRIDRTR KLPVTVLLRALGFSSDQEIIDLLGDNEYLRNTLEKDNTDSTEKALIEIYERLRPGEPPTLENAK NLLASRFFDPKRYDLASVGRYKINKKLHIKNRLFNQRLAETIIDPETKEVIAEAGAMIDRRTLN RLLPYLEKGVGLQTYRPAEGVVDGDISVQTIKIYAPNDPDGEKVINVIGNGFIAEDVKHITPAD IIASISYFFNLLHGVGDTDDIDHLGNRRLRSVGELLQNQFRIGLSRMERVVRERMSIQDTNTIT PQQLINIRPVIAAIKEFFGSSQLSQFMDQTNPLAELTHKRRLSALGPGGLTRERAGFEVRDVHY SHYGRMCPIETPEGPNIGLINSLSTYAKVNKFGFIETPYRRVDPETGRVTDQIDYLTADEEDNY VVAQANVPLAEDGTFLEENVVARFRGENIVVKRDRVDYMDVSPKQVVSAATACIPFLENDDSNR ALMGANMQRQAVPLLEPEAPIVGTGMEYVSAKDSGAAVICKHRGIVERVEAKEIWVRRLIEVDG KEVKGDLDKYRLLKFVRSNQGTCYNQRPIVKKGDIVEKGEILADGPSMDKGELALGRNVLVAFM TWDGYNYEDAIIMSERLVKEDVYTSIHIEEYEAESRDTKLGPEEITRDIPNVGEDALKNLDERG IVRIGAEVKDGDLLVGKVTPKGMTELTAEERLLHAIFGEKAREVRDTSLRVPHGGGIVLDVKVF NREDGDELPPGVNQLVRVYIVQKRKISEGDKMAGRHGNKGVISRILPEEDMPFLPDGTPIDIML NPLGVPSRMNIGQVFELHLGMAAKKLGLHIASPVFDGATEEDVWNILEEAGLARDAKTVLYDGR TGEPFDNRVSVGIMYMIKLAHMVDDKLHARSTGPYSLVTQQPLGGKAQFGGQRFGEMEVWALEA YGAAYTLQEILTVKSDDVVGRVKTYEAIVKGENIPEPGVPESFKVLIKELQSLGMDVTILTSDE QEVNMENFDDDDDHAPDAIMVDVKPAEREEAGEEKDAVTKEGRRAA SEQ ID NO. 3 SGSSMLDVNKFEYMKIGLASPEKIRSWSYGEVKKPETINYRTLKPEKDGLFERIFGPTKDWECH CGKYKRVRYKGVVCDRCGVEVTRSKVRRERMGHIELAAPVSHIWYFKGIPSRMGLVLDMSPRAL EEVIYFASYVVTDPGDTPLEKKQLLSEKEYRAYREKYGQSFQASMGAEAIKKLLQDIDLDKEVA ALKEELKTAQGQRRARIIKRLEVLEAFRSSGNDPAWMVLDVLPVIPPELRPMVQLDGGRFATSD LNDLYRRVINRNNRLKRLLDLGAPNIIVQNEKRMLQEAVDALIDNGRRGRPVTGPGNRPLKSLS HMLKGKKGRFRQNLLGKRVDYSGRSVIVVGPNLKMYQCGLPKEMALELFKPFVMKELVERGLAH NIKSAKRKIERVHPEVWDVLEDVIKEHPVLLNRAPTLHRLGIQAFEPTLVEGRAIRLHPLVCTA YNADFDGDQMAVHVPLSAEAQAEARLLMLAAQNILNPKDGKPVVTPSQDMVLGNYYLTMEREGA IGEGMVFKDTDEALLAYHNGYVHLHSRIAIHAGSLKNETFTEEQNNKLLLTTVGKLIFNEILPN SFPYINEPTTENIEGRTPDKYFLDKGVNVREEIRKRELVPPFKKKVLGQIIAEVFKRFKITETS KMLDRMKDLGFQYSTKAGITIGVADIVVLPEKQEILDEAQAKVDTVLKQFRRGLITDEERYERV ISIWSAAKDKIQDRLMKSLDKRNPIFMMSDSGARGNASNFTQLAGMRGLMANPAGRIIELPIKS SFREGLTVLEYFISTHGARKGLADTALKTADSGYLTRRLVDVAQDVIVREEDCGTDRGILARAL TDGTEVVVKLEERLVGRYAHKTVRHPETGEVIVRKDEMITEDIANEIIKAGITEVWIRSVFACN TRHGVCKKCYGRNMATGMDVEVGEAVGIIAAQSIGEPGTQLTMRTFHTGGVAGDDITQGLPRVQ ELFEARNPKGQAVISEIDGTVISINETRDNQYEIVVQSEVETRTYVAPYNARLKVEEGQRVERG QELTEGSVDPKQLLRVRDITSVQEYLLREVQKVYRMQGVEISDKHIEVMVRQMLRKVRVIDAGD TDVLPGTLLDVHQFTDVNAKALREGKRPATARQVLLGITKASLETDSFLSASFQETTRVLTDA AIKGKRDELLGLKENVIIGKLVPAGTGMARYRKVKPAVKKETAGGTVSSKGRRAA SEQ ID NO. 4 SGSSMLDVNKFEYMKIGLASPEKIRSWSYGEVKKPETINYRTLKPEKDGLFCERIFGPTKDWEC HCGKYKRVRYKGVVCDRCGVEVTRSKVRRERMGHIELAAPVSHIWYFKGIPSRMGLVLDMSPRA LEEVIYFASYVVTDPGDTPLEKKQLLSEKEYRAYREKYGQSFQASMGAEAIKKLLQDIDLDKEV AALKEELKTAQGQRRARIIKRLEVLEAFRSSGNDPAWMVLDVLPVIPPELRPMVQLDGGRFATS DLNDLYRRVINRNNRLKRLLDLGAPNIIVQNEKRMLQEAVDALIDNGRRGRPVTGPGNRPLKSL SHMLKGKKGRFRQNLLGKRVDYSGRSVIVVGPNLKMYQCGLPKEMALELFKPFVMKELVERGLA HNIKSAKRKIERVHPEVWDVLEDVIKEHPVLNRAPTLHRLGIQAFEPTLVEGRAIRLHPLVCTA YNADFDGDQMAVHVPLSAEAQAEARLLMLAAQNILNPKDGKPVVTPSQDMVLGNYYLTMEREGA IGEGMVFKDTDEALLAYHNGYVHLHSRIAIHAGSLKNETFTEEQNNKLLLTTVGKLIFNEILPN SFPYINEPTTENIEGRTPDKYFLDKGVNVREEIRKRELVPPFKKKVLGQIIAEVFKRFKITETS KMLDRMKDLGFQYSTKAGITIGVADIVVLPEKQEILDEAQAKVDTVLKQFRRGLITDEERYERV ISIWSAAKDKIQDRLMKSLDKRNPIFMMSDSGARGNASNFTQLAGMRGLMANPAGRIIELPIKS SFREGLTVLEYFISTHGARKGLADTALKTADSGYLTRRLVDVAQDVIVREEDCGTDRGILARAL TDGTEVVVKLEERLVGRYAHKTVRHPETGEVIVRKDEMITEDIANEIIKAGITEVWIRSVFACN TRHGVCKKCYGRNMATGMDVEVGEAVGIIAAQSIGEPGTQLTMRTFHTGGVAGDDITQGLPRVQ ELFEARNPKGQAVISEIDGTVISINETRDNQYEIVVQSEVETRTYVAPYNARLKVEEGQRVERG QELTEGSVDPKQLLRVRDITSVQEYLLREVQKVYRMQGVEISDKHIEVMVRQMLRKVRVIDAGD TDVLPGTLLDVHQFTDVNAKALREGKRPATARQVLLGITKASLETDSFLSASFQETTRVLTDA AIKGKRDELLGLKENVIIGKLVPAGTGMARYRKVKPAVKKETAGGTVSSKGRRAA SEQ ID NO. 5 SGSSMSLQQQYSPEELQEMSFVELANLILLDKREALPFDQLVREAAALAGISEEEMEARLAQYY TDLNIDGRFICVGENVWGRAWYPFDQTEDETVTIVKPKKKKKALDDEYDEYDELLEDEDLDYDD LDEYDEEELELDEDELLEDEEFDLDEDVAFDDDILGDEEFELGDAPLDEELDLEEPEEDEGRRA A SEQ ID NO. 6 SGSSMLYPSIDLLMQKVDSKYKLVTVVAKRARELQDGAELMVKKPVSKKFVGQALEEIAGDKVE LVEEEKGRRAA SEQ ID NO. 7 MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTMAEKPAQSKQAEAAGESLEQVKEQLAELGKK RGILTYEEIAERLSGFDLDSDQMDEYYEYLAEQGIEVISESDLEADPDIDDLAKEEEFDLNDLS VPPGVKINDPVRMYLKEIGRVPLLSAEEEIELAKRIEQGDEEAKRRLTEANLRLVVSIAKRYVG RGMLFLDLIQEGNMGLIKAVEKFDYRKGYKFSTYATWWIRQAITRAIADQARTIRIPVHMVETI NKLIRVQRQLLQDLGREPTPEEIAEEMDLTPEKVREILKIAQEPVSLETPIGEEDDSHLGDFIE DQDATSPSEHAAYELLKEQLEDVLDTLTDREENVLRLRFGLDDGRTRTLEEVGKVFGVTRERIR QIEAKALRKLRHPSRSKRLKDFLE SEQ ID NO. 8 SGSSMNLVLMGLPGAGKGTQAGKIVEAYGIPHISTGDMFRAAIKEGTPLGLQAKQYMDRGDLVP DELQAKQYMDRGDLVPDEGFLLDGFPRTVAQAEALETLLSEIGRKLDYVIHIDVRQEVLMERLT GRRICRNCGATYHLVFHPPAKPGVCDKCGGDLYQRPDNEATVANRLEVNMKQMKLLLDFYEQKG YLRHINGEQEMEKVFADICEVLGGLARGRRAA SEQ ID NO. 9 SGSSMGQEKLYIEKELSWLSFNERVLQEAADKSNPLIERMRFLGIYSNNLDEFYKVRFAELKRR IIISEEQGSNSHSRHLLGKIQSRVLKADQEFDGLYNELLLEMARNQIFLINERQLSVNQQNWLR HYFKQYLRQHITPILINPDTDLVQFLKDDYTYLAVEIIRGDTIRYALLEIPSDKVPRFVNLPPE APRRRKPMILLDNILRYCLDDIFKGFFDYDALNAYSMKMTRDAEYDLVHEMEASLMELMSSSLK QRLTAEPVRFVYQRDMPNALVEVLREKLTISRYDSIVPGGRYHNFKDFINFPNVGKANLVNKPL PRLRHIWFDKAQFRNGFDAIRERDVLLYYPYHTFEHVLELLRQASFDPSVLAIKINIYRVAKDS RIIDSMIHAAHNGKKVTVVVELQARFDEEANIHWAKRLTEAGVHVIFSAPGLKIHAKLFLISRK ENGEVVRYAHIGTGNFNEKTARLYTDYSLLTADARITNEVRRVFNFIENPYRPVTFDYLMVSPQ NSRRLLYEMVDREIANAQQGLPSGITLKLNNLVDKGLVDRLYAASSSGVPVNLLVRGMCSLIPN LEGISDNIRAISIVDRYLEHDRVYIFENGGDKKVYLSSADWMTRNIDYRIEVATPLLDPRLKQR VLDIIDILFSDTVKARYIDKELSNRYVPRGNRRKVRAQLAIYDYIKSLEQPEGRRAA SEQ ID NO. 10 MRLLPEESWLQFNRRVLRQSERPDFPLLERMRFLAIWNRNLDEFFAARIAKPFLKHRGSAHLRL LKEAEDQARLAEGRYRALLAEAAPHLKILEPEELDDLDWLYFRVFLAEVVVPKTDLIAWEAAKE ASHGALYFASEDHLVRLPQDLPRLLKVPGREGTYVRLGALMRARSDLFLPRESPLYEFRVLRLL ESERARADWDELAQALEGRQEGLSTLLVAERGFPKGWLEGLQEALGLLPEEVILLSPPLNLTLV ETLVAEGPSRWRFPPLEPKRPRAFMKNPLKRLQEKDLVLYHPFEDYAALERFAEAALSEEVEEV YATLYRIGEANPLAEALIQAARAGKRVHVLLEPRARFDELLNLSWYLRFLRAGVAVLPLSERKV HAKALLLLTQKGGYAHLGTGNYNPQNGRQYTDFSLFTARKEVVMEVAEFFRALQEGRTPRLNLL KTGEAIQELLLEHIRAESHPKGRILLKCNHLTDPALLEALARAADKGARVDLIVRSTLTLLHPR FRARSLVGRFLEHARVAAFYQGGRWALYLTSADLMPRNFQNRFELLFPVLDKEGKAKVLEVLKR QLKDDRNAFLLSPKGETPLWGGRHDAQRILAW SEQ ID NO. 11 MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTMRLLPEESWLQFNRRVLRQSERPDFPLLERM RFLAIWNRNLDEFFAARIAKPFLKHRGSEAHLRLLKEAEDQARLAEGRYRALLAEAAPHLKILE PEELDDLDWLYFRVFLAEVVVPKTDLIAWEAAKEASHGALYFASEDHLVRLPQDLPRLLKVPGR EGTYVRLGALMRARSDLFLPRESPLYEFRVLRLLESERARADWDELAQALEGRQEGLSTLLVAE RGFPKGWLEGLQEALGLLPEEVILLSPPLNLTLVETLVAEGPSRWRFPPLEPKRPRAFMKNPLK RLQEKDLVLYHPFEDYAALERFAEAALSEEVEEVYATLYRIGEANPLAEALIQAARAGKRVHVL LEPRARFDELLNLSWYLRFLRAGVAVLPLSERKVHAKALLLLTQKGGYAHLGTGNYNPQNGRQY TDFSLFTARKEVVMEVAEFFRALQEGRTPRLNLLKTGEAIQELLLEHIRAESHPKGRILLKCNH LTDPALLEALARAADKGARVDLIVRSTLTLLHPRFRARSLVGRFLEHARVAAFYQGGRWALYLT SADLMPRNFQNRFELLFPVLDKEGKAKVLEVLKRQLKDDRNAFLLSPKGETPLWGGRHDAQRIA W SEQ ID NO. 12 MPITINNFRYSDPVNNDTIIMMEPPYCKGLDIYYKAFKITDRIWIVPERYEFGTKPEDFNPPSS LIEGASEYYDPNYLRTDSDKDRFLQTMVKLFNRIKNNVAGEALLDKIINAIPYLGNSYSLLDKF DTNSNSVSFNLLEQDPSGATTKSAMLTNLIIFGPGPVLNKNEVRGIVLRVDNKNYFPCRDGFGS IMQMAFCPEYVPTFDNVIENITSLTIGKSKYFQDPALLLMHELIHVLHGLYGMQVSSHEIIPSK QEIYMQHTYPISAEELFTFGGQDANLISIDIKNDLYEKTLNDYKAIANKLSQVTSCNDPNIDID SYKQIYQQKYQFDKDSNGQYIVNEDKFQILYNSIMYGFTEIELGKKFNIKTRLSYFSMNHDPVK IPNLLDDTIYNDTEGFNIESKDLKSEYKGQNMRVNTNAFRNVDGSGLVSKLIGLCKKIIPPTNI RENLYNRTA SEQ ID NO. 13 MHELIHVL SEQ ID NO. 14 SLTDLGGELCIKIKNEDLTFIAEKNSFSEEPFQDEIVSYNTKNKPLNFNYSLDKIIVDYNLQSK ITLPNDRTTPVTKGIPYAPEYKSNAASTIEIHNIDDNTIYQYLYAQKSPTTLQRITMTNSVDDA LINSTKIYSYFPSVISKVNQGAQGILFLQWVRDIIDDFTNESSQKTTIDKISDVSTIVPYIGPA LNIVKQGYEGNFIGALETTGVVLLLEYIPEITLPVIAALSIAESSTQKEKIIKTIDNFLEKRYE KWIEVYKLVKAKWLGTVNTQFQKRSYQMYRSLEYQVDAIKKIIDYEYKIYSGPDKEQIADEINN LKNKLEEKANKAMININIFMRESSRSFLVNQMINEAKKQLLEFDTQSKNILMQYIKANSKFIGI TELKKLESKINKVFSTPIPFSYSKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSS VITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQY GTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVF ITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALN PKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGK LNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRIL RVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNW YFNHLKDKILGCDWYFVPTDEGWTNDGSA SEQ ID NO. 15 ENLYFQS SEQ ID NO. 16 GSGSNGSSGSVSMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPF AWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIY KVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAK KPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYRSGGGHHHHHHGDL SEQ ID NO. 17 MPITINNFRYSDPVNNDTIIMMEPPYCKGLDIYYKAFKITDRIWIVPERYEFGTKPEDFNPPSS LIEGASEYYDPNYLRTDSDKDRFLQTMVKLFNRIKNNVAGEALLDKIINAIPYLGNSYSLLDKF DTNSNSVSFNLLEQDPSGATTKSAMLTNLIIFGPGPVLNKNEVRGIVLRVDNKNYFPCRDGFGS IMQMAFCPEYVPTFDNVIENITSLTIGKSKYFQDPALLLMHELIHVLHGLYGMQVSSHEIIPSK QEIYMQHTYPISAEELFTFGGQDANLISIDIKNDLYEKTLNDYKAIANKLSQVTSCNDPNIDID SYKQIYQQKYQFDKDSNGQYIVNEDKFQILYNSIMYGFTEIELGKKFNIKTRLSYFSMNHDPVK IPNLLDDTIYNDTEGFNIESKDLKSEYKGQNMRVNTNAFRNVDGSGLVSKLIGLCKKIIPPTNI RENLYNRTASLTDLGGELCIKIKNEDLTFIAEKNSFSEEPFQDEIVSYNTKNKPLNFNYSLDKI IVDYNLQSKITLPNDRTTPVTKGIPYAPEYKSNAASTIEIHNIDDNTIYQYLYAQKSPTTLQRI TMTNSVDDALINSTKIYSYFPSVISKVNQGAQGILFLQWVRDIIDDFTNESSQKTTIDKISDVS TIVPYIGPALNIVKQGYEGNFIGALETTGVVLLLEYIPEITLPVIAALSIAESSTQKEKIIKTI DNFLEKRYEKWIEVYKLVKAKWLGTVNTQFQKRSYQMYRSLEYQVDAIKKIIDYEYKIYSGPDK EQIADEINNLKNKLEEKANKAMININIFMRESSRSFLVNQMINEAKKQLLEFDTQSKNILMQYI KANSKFIGITELKKLESKINKVFSTPIPFSYSKNLDCWVDNEEDIDVILKKSTILNLDINNDII SDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPK VSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFN AYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDK FRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLT NAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGN AFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPN RDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDGSAENLYFQSGSGSNGSSGSVSMAIIKEF MRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKH PADIPDYLKLSFPEGFKWERVMNFEDGGVVIVTQDSSLQDGEFIYKVKLRGINFPSDGPVMQKK TMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDIIS HNEDYTIVEQYERAEGRHSTGGMDELYRSGGGHHHHHHGDL SEQ ID NO. 18 ATTCTGCAATCCTATTGTTTATCATACCTGATATATGAAGATACGACACTAGGAGTGA TGGCCATG SEQ ID NO. 19 TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCC SEQ ID NO. 20 CCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACC SEQ ID NO. 21 ATGCCAATAACCATAAATAATTTTAGATATAGTGATCCTGTTAATAATGATACAATTATTATGA TGGAGCCACCATACTGTAAGGGTCTAGATATCTATTATAAGGCTTTCAAAATAACAGATCGTAT TTGGATAGTGCCGGAAAGGTATGAATTTGGGACAAAACCTGAAGATTTTAACCCACCATCTTCA TTAATAGAAGGTGCATCTGAGTATTACGATCCAAATTATTTAAGGACTGATTCTGATAAAGATA GATTTTTACAAACCATGGTAAAACTGTTTAACAGAATTAAAAACAATGTAGCAGGTGAAGCCTT ATTAGATAAGATAATAAATGCCATACCTTACCTTGGAAATTCATATTCCTTACTAGACAAGTTT GATACAAACTCTAATTCAGTATC/TTTTAATTTATCAGAACAAGACCCCAGTGGAGCAACTACA AAATCAGCAATGCTGACAAATTTAATAATATTTGGACCTGGGCCTGTTTTAAATAAAAATGAGG TTAGAGGTATTGTATTGAGGGTAGATAATAAAAATTACTTCCCATGTAGAGATGGTTTTGGTTC AATAATGCAAATGGCATTTTGCCCAGAATATATACCCACTTTTGATAATGTAATAGAAAATATT ACGTCACTCACTATTGGCAAAAGCAAATATTTTCAAGATCCAGCATTACTATTAATGCACGAAC TTATACATGTACTACATGGTTTATACGGAATGCAGGTATCAAGCCATGAAATTATTCCATCCAA ACAAGAAATTTATATGCAGCATACATATCCAATAAGTGCTGAAGAACTATTCACTTTTGGCGGA CAGGATGCTAATCTTATAAGTATTGATATAAAAAACGATTTATATGAAAAAACTTTAAATGATT ATAAAGCTATAGCTAACAAACTTAGTCAAGTCACTAGCTGCAATGATCCCAACATTGATATTGA TAGCTACAAACAAATATATCAACAAAAATATCAATTCGATAAAGATAGCAATGGACAATATATT GTAAATGAGGATAAATTTCAGATACTATATAATAGCATAATGTATGGTTTTACAGAGATTGAAT TGGGAAAAAAATTTAATATAAAAACTAGACTTTCTTATTTTAGTATGAATCATGACCCTGTAAA AATTCCAAATTTATTAGATGATACAATTTACAATGATACAGAAGGATTTAATATAGAAAGCAAA GATCTGAAATCTGAATATAAAGGTCAAAATATGAGGGTAAATACAAATGCTTTTAGAAATGTTG ATGGATCAGGCCTAGTTTCAAAACTTATTGGCTTATGTAAAAAAATTATACCACCAACAAATAT AAGAGAAAATTTATATAATAGAACTGCATCATTAACAGATTTAGGAGGAGAATTATGTATAAAA ATTAAAAATGAAGATTTAACTTTTATAGCTGAAAAAAATAGCTTTTCAGAAGAACCATTTCAAG ATGAAACAGTTAGTTATAATACAAAAAATAAACCATTAAATTTTAATTATTCGCTAGATAAAAT TATTTTAGATTATAATCTACAAAGTAAAATTACATTACCTAATGATAGGACAACCCCAGTTACA AAAGGAATTCCATATGCTCCAAAATATAAAAGTAATGCTGCAAGTACAATAGAAATACATAATA TTGATGACAATACAATATATCAATATTTGTATGCTCAAAAATCTCCTACAACTCTACAAAGAAT AACTATGACTAATTCTGTCGATGACGCATTAATAAATTCCACCAAAATATATTCATATTTTCCA TCTGTAATCAGTAAAGTTAACCAAGGTGCACAAGGAATTTTATTCTTACAGTGGGTGAGAGATA TAATTGATGATTTACCAATGAATCTTCACAAAAAACTACTATTGATAAAATTTCAGATGTATCC ACTATTGTTCCTTATATAGGACCCGCATTAAACATTGTAAAACAAGGCTATGAGGGAAACTTTA TAGGTGCTTTAGAAACTACCGGAGTGGTTTTATTATTGGAATATATTCCAGAAATTACTTTACC AGTAATTGCAGCTTTATCTATAGCAGAAAGTAGCACACAAAAAGAAAAGATAATAAAAACAATA GATAACTTTTTAGAAAAAAGATATGAAAAATGGATTGAAGTATATAAACTAATAAAAGCAAAAT GGTTAGGCACAGTTAATACGCAATTCCAAAAAAGAAGTTATCAAATGTATAGATCTTTAGAATA TCAAGTAGATGCAATAAAAAAAATAATAGACTATGAATATAAAATATATTCAGGACCTGATAAG GAACAAATTGCCGACGAAATTAATAATCTGAAAAACAAACTTGAAGAAAAGGCTAATAAAGCAA TGATAAACATAAATATATTTATGAGGGAAAGTTCTAGATCATTTTTAGTTAATCAAATGATTAA CGAAGCTAAAAAGCAGTTATTAGAGTTTGATACTCAAAGCAAAAATATTTTAATGCAGTATATA AAAGCAAATTCTAAATTTATAGGTATAACTGAACTAAAAAAATTAGAATCAAAAATAAACAAAG TTTTTTCAACACCAATTCCATTTTCTTATTCTAAAAATCTGGATTGTTGGGTTGATAATGAAGA AGATATAGATGTTATATTAAAAAAGAGTACAATTTTAAATTTAGATATTAATAATGATATTATA TCAGATATATCTGGGTTTAATTCATCTGTAATAACATATCCAGATGCTCAATTGGTGCCCGGAA TAAATGGCAAAGCAATACATTTAGTAAACAATGAATCTTCTGAAGTTATAGTGCATAAAGCTAT GGATATTGAATATAATGATATGTTTAATAATTTTACCGTTAGCTTTTGGTTGAGGGTTCCTAAA GTATCTGCTAGTCATTTAGAACAATATGGCACAAATGAGTATTCAATAATTAGCTCTATGAAAA AATATAGTCTATCAATAGGATCTGGTTGGAGTGTATCACTTAAAGGTAATAACTTAATATGGAC TTTAAAAGATTCCGCGGGAGAAGTTAGACAAATAACTTTTAGTGATTTATCTGATAAATTTAAT GCTTATTTAGCAAATAAATGGGTTTTTATAACTATTACTAATGATAGATTATCTTCTGCTAATT TGTATATAAATGGAGTACTTATGAAAAATGCAGAAATTACCGGTTTAGGAGCTATTAGAGAGGA TAATAATATAACATTAAAACTAGATAGATGTAATAATAATAATCAATACGTTTCTATTGATAAA TTTAGGATATTTTGCAAAGCATTAAATCCAAAAGAGATTGAAAAATTATACACAAGTTATTTAT CTATAACCTTTTTAAGAGACTTCTGGGGAAACCCTTTACGATATGATACAGAATATTATTTAAT ACCAGTAGCTTCTAGTTCTAAAGATGTTCAATTGAAAAATATAACAGATTATATGTATTTGACA AATGCGCCATCGTATACTAACGGAAAATTGAATATATATTATAGAAGGTTATATAGTGGACTAA AATTTATTATAAAAAGATATACACCTAATAATGAAATAGATTCTTTTGTTAAATCAGGTGATTT TATTAAATTATATGTATCATATAACAATAATGAGCACATTGTAGGTTATCCGAAAGATGGAAAT GCCTTTAATAATCTTGATAGAATTCTAAGAGTAGGTTATAATGCCCCAGGTATCCCTCTTTATA AAAAAATGGAAGCAGTAAAATTGCGTGATTTAAAAACCTATTCTGTACAACTTAAATTATATGA TGATAAAAATGCATCTTTAGGACTAGTAGGTATCCGTAATGGTCAAATAGGCAACGATCCAAAT AGGGATATATTAATTGCAAGCAACTGGTACTTTAATCATTTAAAAGATAAAACTTTAACATGTG ATTGGTACTTTGTACCTACAGATGAAGGATGGACAAATGATTAA SEQ ID NO. 22 GAAAACCTGTACTTCCAATCCAATATTGGTAGTGGGAGCAACGGCAGCAGCGGATCCGTGAGCA AGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGG CTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACC CAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCC CTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAA GCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTG ACCGTGACCCAGGACTCCTCCCTCCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCA CCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGA GCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGAC GGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCG GCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGA ACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAAGCG GCCGCACTCGAGCACCACCACCACCACCACTGA SEQ ID NO. 23 MERTFVMIKPDGVRRGLVGEILARFERKGFRIAALKLMQISQELAERHYAEHREKPFFPGLVRFITSGPVVAMVLEG PGVVAEVRKMMGATHPKDALPGTIRGDFATTIDENVIHGSATLEDAQREIALFFRPEELL

TABLE 1 Amino Acid Sequence for recombinant TaqPPK showing: 6xHis-Tag; V5 epitope tag and TEV cleavage site (SEQ ID NO. 11) His6 V5 tag TEV \/ TaqPPK MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTMRLLPEESWLQFNRRVL RQSERPDFPLLERMRFLAIWNRNLDEFFAARIAKPFLKHRGSEAHLRLLK EAEDQARLAEGRYRALLAEAAPHLKILEPEELDDLDWLYFRVFLAEVVVP KTDLIANEAAKEASHGALYFASEDHLVRLPQDLPRLLKVPGREGTYVRLG ALMRARSDLFLPRESPLYEFRVLRLLESERARADWDELAQALEGRQEGLS TLLVAERGFPKGWLEGLQEALGLLPEEVILLSPPLNLTLVETLVAEGPSR WRFPPLEPKRPRAFMKNPLKRLQEKDLVLYHPFEDYAALERFAEAALSEE VEEVYATLYRIGEANPLAEALIQAARAGKRVHVLLEPRARFDELLNLSWY LRFLRAGVAVLPLSERKVHAKALLLLTQKGGYAHLGIGNYNPQNGRQYTD FSLFTARKEVVMEVAEFFRALQEGRTPRLNLLKTGEAIQELLLEHIRAES HPKGRILLKCNHLTDPALLEALARAADKGARVDLIVRSTLTLLHPRFRAR SLVGRFLEHARVAAFYQGGRWALYLTSADLMPRNFQNRFELLFPVLDKEG KAKVLEVLKRQLKDDRNAFLLSPKGETPLWGGRHDAQRILAW

TABLE 2 Reaction mixtures of NDPK reactions reflective of the ping-pong mechanism of XDP + YTP -> XTP + TDP ADP/GTP ADP/GTP 4 5× Reaction Buffer 4 5× Reaction Buffer 1 100 mM GTP 0 100 mM GTP 0.5 200 mM ADP 0.5 200 mM ADP 1 5.0 mg/ml NDPK 1 5.0 mg/ml NDPK 13.5 dH2O 14.5 dH2O 20 TOTAL 20 TOTAL ADP/CTP ADP/CTP 4 5× Reaction Buffer 4 5× Reaction Buffer 1 100 mM CTP 0 100 mM CTP 0.5 200 mM ADP 0.5 200 mM ADP 1 5.0 mg/ml NDPK 1 5.0 mg/ml NDPK 13.5 dH2O 14.5 dH2O 20 TOTAL 20 TOTAL ADP/GTP ADP/GTP 4 5× Reaction Buffer 4 5× Reaction Buffer 1 100 mM GTP 1 100 mM GTP 0 200 mM ADP 0.5 200 mM ADP 1 5.0 mg/ml NDPK 0 5.0 mg/ml NDPK 14 dH2O 14.5 dH2O 20 TOTAL 20 TOTAL 

What is claimed is:
 1. A cell-free protein expression system comprising: a bioreactor having: a reaction mixture containing a thermostable Geobacillus cell extract having all cell-free reaction components necessary for in vitro macromolecule synthesis and wherein said Geobacillus is genetically modified to include at least one of the following: knock-out expression of OmpT-homologue; knock-out expression of RNaseI; knock-out expression of DNA-methylation dependent DNase; reduce expression of culture density-dependent sporulation operon; overexpress sigma factor RpoD; and overexpress RNA polymerase (RNAP). at least one nucleic acid synthesis template; a cellular adenosine triphosphate (ATP) energy regeneration system comprising: a quantity of purified Adenosyl Kinase (AdK) enzyme; and a quantity of purified Polyphosphate Kinase (PPK) enzyme; a quantity of inorganic polyphosphate (PPi); and a quantity of adenosine monophosphate (AMP). wherein said AdK and PPK enzymes work synergistically to regenerate cellular ATP energy from PPi and AMP.
 2. The system of claim 1, wherein said Geobacillus comprises Geobacillus stearothermophilus.
 3. The system of claim 1, wherein said cell-free reaction components comprises cell-free reaction components selected from the group consisting of: amino acids; polyphosphate; Tris-Acetate; Mg(OAc)₂; K⁺-glutamate; amino-acetate; NaCl; KCl; MgCl₂; DTT; octyl-b-glycoside; NAD; NADP; sorbitol; FADH; ATP; GTP, UTP, and/or CTP; CoA; PLP; and SAM.
 4. The system of claim 1, wherein said cell-free reaction components comprises cell-free reaction components selected from the group consisting of: 2 mM of each natural amino acid; 1 mg/ml polyphosphate; 5 mM Tris-Acetate; 4 mM Mg(OAc)₂; 12 mM K⁺-glutamate; 1 mM amino-acetate; 100 mM NaCl; 10 mM KCl; 5 mM MgCl₂; 0.1 mM DTT; 0.2% octyl-b-glycoside; 0.8 mM NAD; 0.4 mM NADP; 200 mM sorbitol; 0.5 mM FADH; 1.5 mM ATP; 1 mM GTP, UTP, CTP each; 1 mM CoA; 2 mM PLP; and 0.2 mM SAM.
 5. The system of claim 1, further comprising a quantity of thermostable RNA polymerase (RNAP).
 6. The system of claim 5, wherein said thermostable RNAP comprises a quantity of RNAP derived from Geobacillus stearothermophilus (Gst RNAP).
 7. The system of claim 6, wherein said Gst RNAP comprises RNAP subunits: subunit alpha according to SEQ ID NO. 1; subunit beta according to SEQ ID NO. 2; subunit beta′ according to SEQ ID NO. 3 and/or subunit beta′ (opt) according to SEQ ID NO. 4; subunit delta according to SEQ ID NO. 5; and subunit omega according to SEQ ID NO.
 6. 8. The system of claim 1, further comprising a quantity of tRNAs isolated from the same type of organism used to generate the cell extract.
 9. The system of claim 8, wherein said quantity of tRNAs comprises a quantity of tRNAs isolated from Geobacillus.
 10. The system of claim 1, further comprising a quantity of tRNAs configured to prevent translational stalling due to rare codons.
 11. The system of claim 10, wherein said quantity of tRNAs configured to prevent translational stalling due to rare codons comprises a quantity of tRNAs isolated from E. coli.
 12. The system of claim 10, further comprising a quantity of Sorbitol-dehydrogenase (SDH).
 13. The system of claim 12, wherein said SDH comprises an SDH from Geobacillus stearothermophilus (Gst SDH).
 14. The system of claim 13, further comprising a quantity of sorbitol.
 15. The system of claim 1, wherein said macromolecule comprises a polypeptide.
 16. The system of claim 1, wherein said nucleic acid synthesis template comprises a linear DNA template.
 17. The system of claim 16, wherein said nucleic acid synthesis template comprises a codon optimized nucleic acid synthesis template.
 18. The system of claim 1, wherein said nucleic acid synthesis template comprises nucleic acid synthesis template having: at least one target expression product gene operably linked to a promoter; at least one ribosome binding site (RBS); at least one expression product cleavage site; and at least one tag.
 19. The system of claim 18, wherein said promotor comprises either a RhIII promotor according to SEQ ID NO. 18, or a T7 promotor according to SEQ ID NO.
 19. 20. The system of claim 18, wherein said at least one target expression product gene operably linked to a promoter comprises a tentoxilysin (TetNT) gene operably linked to a promoter.
 21. The system of claim 20, wherein said TetNT gene comprise a recombinant a first construct coding for the TetNT light chain according to SEQ ID NO. 12, with an autocatalytic protease site according to SEQ ID NO. 13 and disulfide bridge forming cysteines, and a second construct coding for the TetNT heavy chain according to SEQ ID NO. 14, containing a partially integrated TEV site according to SEQ ID NO. 15, and mCherry-His6 according to SEQ ID NO.
 16. 22. The system of claim 20, wherein said TetNT gene comprises a recombinant TetNT gene according to SEQ ID NO.
 21. 23. The system of claim 22, wherein said RBS comprises an RBS according to SEQ ID NO.
 20. 24. The system of claim 23, wherein said expression product cleavage site comprises a TEVsite according to SEQ ID NO.
 15. 25. A The system of claim 18, wherein said a target expression product is a peptide according to SEQ ID NO.
 17. 26. The system of claim 1, wherein said quantity of purified AdK enzyme comprises a quantity of purified thermostable AdK enzyme.
 27. The system of claim 26, wherein said quantity of purified thermostable AdK enzyme comprises a quantity of purified thermostable AdK enzyme derived from Geobacillus stearothermophilus (Gst Adk).
 28. The system of claim 27, wherein said Gst Adk enzyme comprises a peptide according to SEQ ID NO.
 8. 29. The system of claim 1, wherein said quantity of purified PPK enzyme comprises a quantity of purified thermostable PPK enzyme.
 30. The system of claim 29, wherein said quantity of purified thermostable PPK enzyme comprises a quantity of purified thermostable PPK enzyme derived from Thermus aquaticus (TaqPPK).
 31. The system of claim 30, wherein said TaqPPK enzyme comprises a peptide according to SEQ ID NO.
 11. 32. The system of claim 1, wherein said quantity of purified AdK enzyme and said quantity of purified PPK enzyme comprises a quantity of purified Gst AdK enzyme according to SEQ ID NO. 8, and a quantity of purified TaqPPK enzyme according to SEQ ID NO.
 11. 33. The system of claim 32, wherein said quantity of Gst Adk enzyme is greater than said quantity of TaqPPK enzyme.
 34. The system of claim 33, wherein said molar ratio of Gst Adk:TaqPPK is approximately 3:1.
 35. The system of claim 1, wherein said quantity of PPi comprises a PPi concentration range that maintains the equilibrium of the ATP regeneration reaction.
 36. The system of claim 35, wherein said quantity of PPi comprises a PPi concentration range that maintains the equilibrium of the ATP regeneration reaction comprises a concentration range of approximately 0.2-2 mg/ml PPi in a 100 μl volume ATP regenerating reaction.
 37. The system of claim 1, wherein said cellular ATP energy regeneration system components are added to said cell-free expression bioreactor as batch, continuous flow, or semi-continuous flow.
 38. The system of claim 1, wherein said cell-free reaction components are added to said cell-free expression bioreactor as batch, continuous flow, or semi-continuous flow.
 39. An inorganic polyphosphate-based energy regeneration system comprising: an ATP-dependent system supplemented with the following: a quantity of purified Adenosyl Kinase (AdK) enzyme; and a quantity of purified Polyphosphate Kinase (PPK) enzyme; a quantity of inorganic polyphosphate (PPi); and a quantity of adenosine monophosphate (AMP); wherein said quantity of AdK enzyme comprises a quantity of AdK enzyme derived from Geobacillus stearothermophilus (Gst Adk) according to SEQ ID NO. 8, and/or said quantity of PPK enzyme comprises a quantity of PPK derived from Thermus aquaticus (TaqPPK) according to SEQ ID NO.
 11. 40. The system of claim 39, wherein said quantity of Gst Adk enzyme is greater than the quantity of TaqPPK enzyme.
 41. The system of claim 40, wherein said molar ratio of Gst Adk:TaqPPK enzyme is approximately 3:1.
 42. The system of claim 41, wherein said quantity of PPi comprises a concentration range that maintains the equilibrium of the ATP regeneration reaction.
 43. The system of claim 42, wherein said quantity of PPi comprises a concentration range that maintains the equilibrium of the ATP regeneration reaction comprises a concentration range of approximately 0.2-2 mg/ml PPi in a 100 μl volume ATP regenerating reaction.
 44. A cell-free protein expression system comprising: a cell-free expression bioreactor having: a reaction mixture containing a cell extract having all cell-free reaction components necessary for in vitro macromolecule synthesis; at least one nucleic acid synthesis template; a quantity of sorbitol-dehydrogenase (SDH) from Geobacillus stearothermophilus (Gst SDH); a quantity of sorbitol; a cellular adenosine triphosphate (ATP) energy regeneration system comprising: a quantity of purified Adenosyl Kinase (AdK) enzyme; and a quantity of purified Polyphosphate Kinase (PPK) enzyme; a quantity of inorganic polyphosphate (PPi); and a quantity of adenosine monophosphate (AMP). wherein said AdK and PPK enzymes work synergistically to regenerate cellular ATP energy from PPi and AMP.
 45. The system of claim 50, wherein said cell extract comprises a thermophilic bacterial cell extract and/or a thermostable bacterial cell extract.
 46. The system of claim 51, wherein the bacterial cell extract comprises a Geobacillus cell extract.
 47. The system of claim 52, wherein said Geobacillus cell extract comprises a Geobacillus stearothermophilus cell extract.
 48. The system of claim 46, wherein said Geobacillus is genetically modified to include at least one of the following: knock-out expression of OmpT-homologue; knock-out expression of RNaseI; knock-out expression of DNA-methylation dependent DNase; reduce expression of culture density-dependent sporulation operon; overexpress sigma factor RpoD; and overexpress RNA polymerase (RNAP), or any combination thereof. 